Methods and Means for Metabolic Engineering and Improved Product Formation by Micro-Organisms

ABSTRACT

The invention relates to the field of biochemistry, molecular biology and microbiology. More specific the invention relates to methods and means for metabolic engineering and improved product formation by a filamentous micro-organism or a low G+C gram-positive bacterium. 
     The invention discloses that DasR and DasR binding sites play an important and universal role in the control of gene expression in micro-organisms Based on this finding, the invention provides multiple useful applications, such as a method for regulating the expression of a gene of interest, a method for controlling metabolism, a method for decreasing undesired expression and many more. Moreover, the invention also provides means that can be used to establish said methods: for example a micro-organism in which the DasR binding site in operable linkage with a particular gene has been modified to obtain increased or decreased expression of a protein (being a desired or undesired protein) encoded by said gene.

CROSS-REFERENCE TO RELATED APPLICATION

This is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/NL2007/050061, filed Feb. 14, 2007, published in English as International Patent Publication WO 2007/094667 Al on Aug. 23, 2007, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 06075336.5, filed Feb. 14, 2006, the entire disclosure of each of which is hereby incorporated herein by this reference.

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TECHNICAL FIELD

The invention relates to the field of biochemistry, molecular biology and microbiology. More specifically, the invention relates to methods and means for metabolic engineering and improved product formation by a filamentous microorganism or a low G₊C gram-positive bacterium.

BACKGROUND

Filamentous micro-organisms are widely used as industrial producers of products such as antibiotics, anticancer agents, antifungicides and enzymes (Bennett, 1998; Demain, 1991; Hopwood et al., 1995). These organisms include the eukaryotic filamentous fungi (ascomycetes) and the prokaryotic actinomycetes (e.g. Amycolatopsis, Nocardia, Thermobifido and Streptomyces). The market capitalization for antibiotics and enzymes totals around 28 and 2 billion dollars per year, respectively. The soil-dwelling streptomycete Streptomyces coelicolor constitutes an important model system for the study of bacterial development and antibiotic production (Locci, 1986). Streptomyces colonies form a meshwork of vegetative mycelia from which aerial, spore-forming hyphae differentiate (Chater, 1998). Their morphogenesis is controlled by a complex, spatial and temporal genetic programming scheme that is switched on upon nutrient limitation (Schauer et al., 1988; Willey et al., 1991). Streptomycetes are principal protagonists in the recycling and mineralization of organic compounds of dead plants, fungi and insects, which are composed of the polysaccharides cellulose, xylan, and chitin, the most abundant carbon sources on earth (Hodgson, 2000). Hence, they also play a crucial role in our hunt for renewable sources. Interestingly, the study of genome sequences of actinomycetes has unveiled a surprisingly large number of cryptic antibiotic biosynthesis clusters and novel enzymes with industrial potential, thus offering new challenges for directed discovery of natural products, including drugs and enzymes (Hopwood, 2003). For example, a novel screening technique established that selective growth conditions can induce the normally dormant biosynthetic clusters for enediyne-type anti-tumour antibiotics (Zazopoulos et al., 2003).

Global regulation in bacteria involves the presence of pleiotropic-acting transcription factors that coordinate expression of genes, operons and regulons of diverse cellular processes (Martinez-Antonio and Collado-Vides, 2003). Escherichia coli has seven global transcription factors that together regulate approximately half of its genes. The most prominent one is the cyclic AMP receptor protein Crp directly controlling around 200 target genes (Bruckner and Titgemeyer, 2002; Gosset et al., 2004; Zhang et al., 2005). Crp represents the paradigm of a genetic regulator, its properties having attained textbook status and the Crp-cAMP mediated regulation of alternative carbon sources in E. coli is probably the most classical example to illustrate mechanisms that modulate genes expression. Uncovering such pleiotropic regulators is crucial for our understanding of the life style of bacteria and since the elucidation of the role of Crp in carbon catabolite repression (CCR), scientists devoted to carbon utilization always refers to this model to discuss the situation in other micro-organisms. Hence, the catabolite control protein CcpA is a similarly global regulator in low G+C Gram-positive bacteria, and controls more than 300 genes in Bacillus subtilis (Moreno et al., 2001; Titgemeyer and Hillen, 2002). CcpA controls genes involved in CCR, glycolysis, nitrogen assimilation, and phosphate metabolism (Bruckner and Titgemeyer, 2002).

So far, the study of carbon utilization in streptomycetes failed to discover a global regulator, and only resulted in examples of specific regulators controlling individual sugar regulons (Hindle and Smith, 1994; Parche et al., 1999; van Wezel et al., 1997). Interestingly, streptomycetes privileged another category of regulator/sensor element to globally mediate the shift from CCR to substrate induction, using MsiK as the master switch to provide energy to many sugar-specific ABC transporters (Hurtubise et al., 1995; Schlosser et al., 1997; Schlosser et al., 1999; Schlosser, 2000). However, a conserved regulatory motif identified upstream of few genes related to carbohydrate metabolism was still intriguing the research community devoted to carbon regulation, and maintained the idea that perhaps a global regulator would exist (Nothaft et al., 2003; Rigali et al., 2004; Studholme et al., 2004). Our in silico analysis of the helix-turn-helix GntR family (Rigali et al., 2002) recently identified new cis/trans regulatory codes that predict that DasR (SCO5231) regulates the phosphotransferase system specifically for the uptake of N-acetylglucosamine (PTS^(Nag)) (Rigali et al., 2004).

DISCLOSURE OF THE INVENTION

The present inventors disclose that the protein DasR is a regulatory master switch that is involved in multiple processes in micro-organisms.

In a first embodiment, the invention provides a method for identifying a gene which expression is controlled by a DasR-like protein, comprising comparing the DasR binding site consensus sequence with a candidate sequence and selecting the candidate sequence that has a matrix score of at least 5.0.

DasR proteins show a high homology in actinomycetes. All of these DasR proteins comprise a DNA binding motif, preferably a helix-turn-helix DNA binding motif. FIG. 14 shows an alignment of DasR homologues of actinomycetes. The DNA binding motif lies in roughly the first 80 amino acids of the protein (the most important signature is indicated by HTH, for Helix-turn-helix domain, in FIG. 14). FIG. 20 presents an alignment of DasR binding motifs from 10 different streptomycetes. The second part of the protein (roughly 80-255) has homology to the effector binding domain (EBD) of other GntR-type transcriptional regulators, and is therefore the place where binding of glucosamine-6-P is expected to take place.

A DasR-like protein is typically a DasR protein or a functional equivalent and/or a functional fragment thereof, preferably obtained/derived from an actinomycete, which DasR protein comprises the above outlined characteristics, i.e. comprising an DNA binding domain, an EBD and for example capable of binding to a (consensus) DasR binding site. Examples of DasR proteins are the DasR proteins presented in FIG. 14 (A or B or C). It is clear for the skilled person that for example a DasR protein from Streptomyces coelicolor can be modified without significantly changing the above outlined characteristics, for example, by introducing point mutations or (small) deletions. Hence, a DasR-like protein is a DasR protein, preferably obtained from for example Streptomyces coelicolor, Streptomyces clavuligerus, Streptomyces avermitilis, Streptomyces griseus, Streptomyces scabies, Streptomyces species 139 or Thermobifido fusca, possibly comprising mutations which do not interfere significantly with for example the binding of said DasR-like protein to a (consensus) DasR binding site (or DasR target site; the terms will be used interchangeable herein). The Streptomyces clavuligerus DasR sequence has been determined by the present inventors and the nucleic acid sequence as well as the amino acid sequence is depicted in FIG. 19. Hence, in yet another embodiment the invention provides an isolated or recombinant nucleic acid encoding the protein as depicted in FIG. 19B. In a preferred embodiment said nucleic acid is the nucleic acid as depicted in FIG. 19A.

To establish the degree of variation between the DNA binding motifs of DasR in various other streptomycetes, we determined the DNA sequence of the corresponding part of dasR from several other streptomycetes, and derived the amino acid sequence (FIG. 20). This shows that the DNA binding motif of Streptomyces dasR genes and their gene products are very highly conserved, and will bind the same binding sites in all streptomycetes. Therefore, in another preferred embodiment, the DNA binding motif of DasR matches that of the consensus sequence provided in FIG. 20.

It is clear from FIGS. 14A or B that a DasR-like protein cannot only be derived from an actinomycete, but also from a low G+C gram-positive bacteria, such as a Streptococcus. Moreover, from FIG. 14C it is clear that a method or a means according to the invention can also be performed/provided with a DasR homologue that was identified in Streptomyces coelicolor. Of course any suitable combination of these DasR-like proteins can also be made. Hence, the term DasR-like protein comprises not only a DasR protein from an actinomycete but also of a lower G+C gram-positive bacterium or a DasR homologue identified in any of the mentioned organisms.

The consensus sequence for the DasR binding site (dre) in actinomycetes is NN(T/A)GG(T/A)(C/G)T(A/G)N(A/T)C(C/A)(A/C)N (SEQ ID NO:1), where the most highly conserved nucleotides (occurring in more than 80% of all known DasR binding sites) are underlined. In a preferred embodiment, the consensus sequence is (A/T)N(T/A)GGTCTANACCAN (SEQ ID NO:2). In an even more preferred embodiment, the DasR binding site in actinomycetes is ACTGGTCTACACCA(G/C) NO:3. N can be any nucleotide (G, A, T or C) and two nucleotides between parentheses, for example (T/A) means that one of the mentioned nucleotides is present. In this particular example: T or A.

The consensus sequence for the DasR binding site in actinomycetes is also used to find sites in other bacteria, and preferably in other Gram-positive bacteria. The identified consensus sequence for Bacillus species is (A/G)N(T/A)(G/T)(G/A)T(C/A)TA(G/T)A(C/T)(C/A)(A/T)N(T/C) NO:4, that for Lactococcus species is A(T/A)(T/C)(G/A)(G/A)TATATA(C/T)(C/T)(A/G)(A/T)T NO:5, that for Listeria species is A(T/C)(T/C)(G/T)(G/A)T(A/C)TA(T/G)A(C/T)(C/A)(A/G)(A/G)T NO:6, and that for Streptococcus species is (A/T)(T/A)T(G/A)(G/T)(A/C)TA(T/G)N(C/A)(C/T)A(A/T)(T/A) NO:7. Again, N can be any nucleotide (G, A, T or C) and two nucleotides between parentheses, for example (A/G) means that one of the mentioned nucleotides is present. In this particular example: A or G. The invention thus provides a method for identifying a gene which expression is controlled by a DasR-like protein, comprising comparing the DasR binding site consensus sequence with a candidate sequence and selecting the candidate sequence that has a matrix score of at least 5.0, wherein said gene is present in an actinomycete, a bacillus, a lactococcus, a listeria or a streptococcus, preferably in combination with the corresponding identified DasR binding site consensus sequence.

Preferably, a candidate gene which expression is controlled by a DasR-like protein is determined by using a position weight matrix. The matrix score is determined according to the method described by Rigali et al. 2004 and in the materials and methods section of the present invention. In brief, a set of known DasR binding sites is used to perform a first search in the target organism, e.g. Streptomyces coelicolor. This results in new sites, which are experimentally validated. The obtained known targets are used to build a position weight matrix and this is used to predict the complete regulon of the target organism.

In a preferred embodiment, the candidate sequence has a matrix score of at least 7.0. Even more preferred is a score equal or higher than 9.0. All targets with a score above 9 that have been tested were found to be bound by DasR.

In yet an even more preferred embodiment, a method according to the invention further comprises testing whether the expression of said selected candidate sequence is indeed controlled by DasR. This is for example accomplished by testing the binding of a DasR-like protein to said selected identified candidate sequence.

The DasR binding sites are typically located upstream of the gene of interest. Table 2 shows the location of the DasR binding sites relative to the translational start site of the genes of interest. More specifically, in a non-limiting explanation, if the DasR binding site is located upstream of the promoter of the gene of interest, it is likely that it has an activating role on the transcription of said gene of interest. Conversely, if the DasR binding site overlaps the promoter or is located downstream of it, it is likely that it functions as a repressor of the transcription of said gene of interest. However, some proven DasR binding sites have been found inside a gene of interest. One example of this is the target gene malX2 of S. olivaceoviridis (Genbank Q8GBT8). In this case, the dre site (ACTGGTCTACACCACC) (SEQ ID NO:8) is almost identical to that of S. coelicolor malX2 (ACTGGTCTACACCAGT (SEQ ID NO:9); #3 in Table 2, matrix score of 16.2), but located between nt positions +36 and +51 downstream of the translational start site. Binding of DasR to this site was proven by EMSA on a double-stranded oligonucleotide. Therefore, it is clear that a DasR binding site can be functional both upstream and inside of a gene of interest.

With the above-described method, the present inventors have established (by using a novel and restrictive DasR binding site position weight matrix) that more than 200 genes are directly controlled by DasR, including regulons for central and secondary metabolism. These genes are derived from around 130 transcription units that are described in Table 2. The identified genes belong to different categories or kinds of genes, such as genes related to sugar or aminosugar metabolism or genes involved in polysaccharide degradation (summarised in FIG. 1B). The list of DasR targets also includes transcription factors and hence it is concluded that DasR is also involved in indirect transcriptional control. The diversity of genes that is controlled/regulated by DasR is large and hence the present inventors consider DasR to be a master switch in the regulation of gene expression in micro-organisms.

Now that the inventors have disclosed that DasR and DasR binding sites play such an important and universal role in the control of gene expression in micro-organisms, the invention provides multiple useful applications, such as a method for regulating the expression of a gene of interest, a method for controlling metabolism, a method for decreasing undesired expression and many more. Moreover, the invention also provides means that can be used to establish said methods: for example a micro-organism in which the DasR binding site in operable linkage with a particular gene has been modified to obtain increased or decreased expression of a protein (being a desired or undesired protein) encoded by said gene. Other examples of methods and means will be provided in the following description.

In one embodiment, the invention provides a method for regulating the expression of a gene of interest in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with said gene of interest or a DasR-binding site in operable linkage with a gene whose product is involved in the expression of said gene of interest.

Said method for regulating the expression of a gene of interest may be a method for increasing the expression of a gene of interest or a method for decreasing the expression of a gene of interest. This is, amongst others, dependent on whether expression of said gene of interest is desired or undesired. In case expression of a gene of interest is desired, expression is preferably increased and in case expression of a gene of interest is not desired (i.e. is undesired), expression is preferably decreased. Whether the action of said compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site must result in increased or decreased binding between said protein and said site, depends on whether expression of said gene of interest is, upon binding of a DasR-like protein to a DasR binding site, repressed or activated. It is clear from Table 2 (as well as from FIG. 4), that the expression of some genes is repressed upon binding of a DasR-like protein to a DasR-binding site (for example #1, 3, 5, or 7 of Table 2) and that expression of some other genes is activated upon binding of a DasR-like protein to a DasR-binding site (for example #13 of Table 2). All the necessary information to decide the right course of action is presented in the present application. Moreover, information on whether expression of a particular gene or operon is activated or repressed is expanded by using micro-array approaches. One example of such an experiment is the comparison of the expression of genes in a DasR-like protein comprising micro-organism to the expression of genes in a DasR-like protein (null) mutant comprising micro-organism. Another example is a comparison of the expression of genes in a DasR-like protein comprising micro-organism grown in the presence or absence of an inducer molecule (e.g. glucosamine-6-P). Yet another example is the use of the so-called ChIP-on-chip approaches, where DasR-like protein bound DNA fragments are hybridized to microarrays. This will identify the targets directly bound by DasR.

As will be discussed in more detail later, a DasR-like protein comprising micro-organism is a micro-organism that comprises a DasR-like protein or an organism that is capable of expressing said DasR-like protein from a nucleic acid encoding said protein. Moreover, said micro-organism may be an organism that is by nature capable of expressing said DasR-like protein or it may be a micro-organism which has been genetically modified to produce DasR-like protein. Moreover, it is also possible to provide a micro-organism that is already capable of expressing (endogenous) DasR-like protein with a further nucleic acid that enables production of more DasR-protein. Such an additional nucleic acid encoding a DasR-like protein may encode the endogenous DasR or it may encode a heterologous DasR-like protein.

As already outlined above the compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site may result in increased or decreased binding of a DasR-like protein to said binding site, i.e. interfering must be understood to read on increasing the binding as well as on decreasing the binding between said protein and said binding site.

As also already outlined above, DasR-regulated genes are very diverse and may range from a gene involved, in for example, a metabolic process as well as a gene which is a transcription factor. Moreover, a lot of processes in an organism involve so-called cascades of (enzyme) reactions. Hence, the DasR-binding site may be in operable linkage with a particular gene of interest (i.e. the direct end product) or the DasR-binding site may be in operable linkage with a gene whose product is involved in the expression of said gene of interest (for example an intermediate in a cascade or a transcription factor involved in the gene expression of a gene of interest). Hence, a method according to the invention is capable of directly regulating expression of a gene of interest or a method according to the invention is capable of indirectly regulating expression of a gene of interest.

Direct regulation includes a situation in which the gene of interest is a single transcription unit or locus, or where the gene of interest is part of an operon.

Indirect regulation also includes a situation in which a DasR-like protein regulates the expression of a regulator (protein or nucleic acid) whereby the regulator is capable of regulating expression of another gene. Said regulator may be responsible for regulating expression of a target gene or a target operon or a target regulon. This part of the invention is supported by the finding that Table 2 also includes 16 transcription factors (#27, 31, 46, 51, 56, 62, 67, 71, 77, 81, 84, 105, 119, 129, 130 and 131), supporting an extensive level of indirect transcriptional control by DasR.

There are multiple ways in which a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site may be provided to a micro-organism. This is also dependent on the type of compound (which will be discussed in more detail hereunder). If the compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site is for example a small and/or chemical compound which is taken up (for example via endocytosis or fagocytosis or actively transported across the membrane) by said micro-organism, said compound is simple added to the surroundings (for example growth medium) of said micro-organism. If the compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site is a nucleic acid (DNA or RNA), said micro-organism is preferably provided (for example via transfection or transduction or transformation) with said nucleic acids with any of the well known techniques therefore.

As already mentioned the compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site may be very diverse in nature, for example a small (chemical) molecule, metal or ion, a signalling molecule such as a γ-butyrolactone or of peptide origin, a non-ribosomal peptide, a protein or a nucleic acid. In one of the preferred embodiments, said compound is a genetic compound, i.e. a nucleic acid (DNA or RNA). One example of a suitable nucleic acid is a dasR nucleic acid, i.e. a nucleic acid encoding a DasR-like protein (or a functional fragment or derivative thereof). Even more preferably, such a dasR nucleic acid is under control of a strong or inducible promoter. If expression of a gene of interest in operable linkage with a DasR-binding site is normally activated upon binding of a DasR-like protein, the presence of more (for example over-expressed DasR) is capable of further inducing/increasing/activating expression of said gene of interest. If expression of a gene of interest in operable linkage with a DasR-binding site is normally repressed upon binding of a DasR-like protein and if expression of said gene of interest results in an undesired product, the presence of more (for example over-expressed DasR) is capable of further reducing/decreasing/repressing expression of said gene of interest and the amount of undesired product is at least in part (further) decreased. A second example of a suitable nucleic acid is a nucleic acid encoding a mutant DasR-like protein, for example a DasR-like protein with reduced binding capacity for a DasR-binding site or a DasR-like protein with improved binding capacity for a DasR-binding site or a DasR-like protein that is no longer capable of being activated by a so-called inducer (for example a DasR-like protein mutated in or lacking an effector binding domain). Based on the information provided in FIG. 14, a skilled person can easily determine which part of a DasR protein can be modified to obtain a certain result. For example, mutations (deletions, insertion, (point)mutations) in the DNA binding part will have an effect on the binding capacity of such a modified DasR-like protein to a DasR-binding sequence. The effect of provided mutations are easily tested by a binding assay. A third example of a suitable nucleic acid is a nucleic acid containing a modified DasR-binding site (either with decreased of increased DasR-protein binding capacity). Preferably such a nucleic acid is placed into the genome of a particular micro-organism in such a way that the DasR-binding site normally present in front of a gene of interest is exchanged with the modified binding site. Moreover, a gene of interest whose expression is not regulated by DasR can be modified to include a (modified) DasR-binding site upstream of or inside the gene. Optionally, the relevant micro-organism is further provided with DasR-like protein (as a protein or as a nucleic acid encoding said DasR-like protein) and the expression of said gene is now controlled by DasR. The use of a (modified) DasR-binding site typically involves a homologous recombination process. Techniques to obtain homologous recombination are known by the skilled person and hence no further details are provided on said subject matter. A fourth example of a suitable nucleic acid is a nucleic acid that represents a modified dasR promoter. The endogenous expression of DasR is autoregulated. If DasR protein is present in certain amounts it will auto-repress its own expression until the amount of DasR is below a certain threshold level. Modifying the DasR-promoter to a promoter which is not autoregulated increases the amount of available DasR protein and hence especially expression of a gene of interest which is increased upon binding of a DasR-like protein to a DasR binding site in operable linkage with said gene is increased. It is clear to a skilled person that the use of such a nucleic acid preferably involves a recombination process in which the originally present dasR promoter is (at least partly) exchanged for the modified dasR promoter. A fifth example of a suitable nucleic acid is related to the third example and comprises a nucleic acid which arranges for an additional or a deleted DasR binding site. The introduction of additional DasR binding sites in operable linkage with a gene of interest is especially useful in cases of gene expression which is activated upon DasR binding and the deletion of a DasR binding site is for example useful in cases of gene expression that are repressed upon DasR binding but whose expression is desired.

Yet another example of a very useful compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site is an inducer. Such an inducer is very useful in case the expression of a gene of interest is repressed upon binding of a DasR-like protein to a DasR binding site in operable linkage with a gene of interest. Preferably, such an inducer is a derivative of a sugar. As will be disclosed within the experimental part, glucosamine-6-phosphate (the term glucosamine-6-P will be used interchangeably herein) is a very potent inducer of DasR-repressed gene expression. In a preferred embodiment, N-acetylglucosamine (or a derivative or a multimer thereof) is added to the environment of a micro-organism and said N-acetylglucosamine will be converted upon transport into said micro-organism, to glucosamine-6-phosphate. The use of such an inducer is extremely advantageous, because it does not involve any genetic manipulation of the micro-organism at all, but it simple involves manipulation from the outside (i.e. addition of N-acetylglucosamine or a derivative or a multimer thereof).

The present inventors have identified two possible transporters for N-acetylglucosamine, namely the adjacent genes nagE1 and nagE2. As described in the experimental part, mutants of these genes have been constructed and it turns out that nagE2 is the gene encoding a transporter for N-acetylglucosamine. This finding provides even more examples of useful compounds capable of interfering with the binding of a DasR-like protein to a DasR-binding site. For example, if a DasR-like protein comprising micro-organism as used in any of the methods of the invention is incapable of producing (functional) NagE2, said micro-organism is not capable of responding to externally provided and/or formed/produced N-acetylglucosamine (GlcNAc) and DasR will stay active (in case of a DasR repressed gene, DasR remains bound). In another example, the amount of GlcNAc transporter is increased, this is for example obtained by providing a micro-organism with a nucleic acid (preferably nagE2) encoding said GlcNAc transporter. In this case more GlcNAc will be transported into a cell and as a consequence more glucosamine-6-phosphate will be produced. As a result DasR-repressed expression will be decreased and expression of a gene which expression is normally repressed will be increased. In yet another example, the gene encoding the GlcNAc -transported is mutated such that only a variant of GlcNAc is transported into the cell.

Yet another example of a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site is the use of an antibody (or a fragment thereof, for example the binding part) that binds to either DasR or to the DasR binding site and thus at least in part blocks the binding of DasR to a DasR binding site.

It is clear to the skilled person, that any of the above-mentioned compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site may optionally be combined.

As already described the kind of genes whose expression is regulated by DasR is very diverse. For a summary of the different categories of genes regulated by DasR see also FIG. 1B. One group of such genes, are genes that are involved in metabolic processes. Therefore, in yet another embodiment, the invention provides a method for controlling metabolism in a DasR-like protein comprising micro-organism, comprising regulating the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene whose product is part of a metabolic route. In a preferred embodiment, the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene whose product is part of a metabolic route is regulated by providing to said micro-organism a compound capable of interfering with the binding of said DasR-like protein to said DasR-binding site.

It is clear that the compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene whose product is part of a metabolic route may be very diverse in nature (as already discussed above), for example a small (chemical) molecule, metal or ion, a signalling molecule such as a γ-butyrolactone or of peptide origin, a non-ribosomal peptide, or a protein or a nucleic acid. In one of the preferred embodiments, said compound is a genetic compound, i.e. a nucleic acid. Examples of suitable nucleic acids are already given above. In case expression of such a metabolic gene is decreased upon binding of DasR protein to a DasR binding site in operable linkage with said gene, it is also possible (as discussed above) to use an inducer, such as glucosamine-6-phosphate.

Depending on whether expression of said particular gene whose product is part of a metabolic route (or involved in metabolism) is repressed or activated upon binding of DasR to a DasR binding site in operable linkage with said particular gene, the skilled person is capable (based on the herein given guidance) to decide if the binding between DasR protein and a DasR binding site in front of said particular gene must be increased or decreased and moreover, the application provides multiple example of how to increase or decrease binding.

Examples of genes whose product are part of a metabolic route are not-limiting mentioned in Table 2. One example relates to genes involved in glutamate/glutamine metabolism. These are summarised in Table 4. These genes encode enzymes involved in the synthesis or degradation of Glu/Gln, but also tRNA molecules required for the translation of codons that specify incorporation of a Glu or Gln amino acid into a polypeptide synthesized by the ribosome.

A second example involves the metabolism of N-acetylglucosamine and related molecules. The pathway for N-acetylglucosamine is linked to that of glutamate, as shown in FIG. 5. Examples include the chitinolytic genes that convert the polymer chitin into N-acetylglucosamine (e.g. #7, 11, 12, 14, 19, 23, 25, 26 in Table 2), the PTS that transports N-acetylglucosamine into the cell (e.g. #1, 2, 3, 5 in Table 2), and the metabolic genes nagK, nagA (#10, operon) and nagB (#15).

Another nucleic acid relating to glutamate and N-acetylglucosamine metabolism is an RNA ribozyme, which processes the glmS gene in Bacillus subtilis (Winkler et al., 2004), and therefore predictable in other gram-positive bacteria such as actinomycetes. The glmS gene is a target of regulation by DasR (#86 in Table 2), and its gene product GlmS uses glutamine and fructose-6-P to form glucosamine-6-P. Processing of the glmS transcript is required for activation of the mRNA. Excitingly, the ribozyme is activated by glucosamine-6-P, which is the metabolic product of the GlmS enzyme. We have shown here that glucosamine-6-P is also the inducer molecule of DasR. Additional data by Winkler et al. showed that the ribozyme responds to the metabolic state and represses the glmS gene in response to rising glucosamine-6-P concentrations.

A third example relates to energy provision, such as enzymes involved in glycolysis (e.g. phosphofructokinase, #74 in Table 2; see also fructose-1,6-bisphosphatase, fructose-1-phosphate kinase and phosphoenolpyruvate synthase in Table 8) and acetyl-CoA formation or processing (e.g. acetoacetyl-CoA synthetase, #12 Table 2; acyl-CoA dehydrogenase, #25 in Table 2; and acetoacetyl-CoA thiolase (ThiL; activated by DasR; FIG. 4). Acetyl CoA is a crucial starting compound of the TCA cycle. Surprisingly, DasR also controls energy provision directly via the control of the nuo operon encoding the subunits of NADH dehydrogenase (#104 in Table 2; two DasR binding sites). This enzyme is the first of the electron transport (redox) chain.

Many of the examples mentioned for Table 2 are also part of Tables 4-7 and 9 that refer to target genes occurring in low G+C Gram-positive bacteria. Therefore, the metabolic engineering approach also applies in a similar fashion to this important class of bacteria, which includes Bacillus, Lactococcus, Lactobacillus, Streptococcus and Listeria.

The metabolic processes or routes that may be influenced according to a method of the invention are amino acid metabolism, peptidoglycan degradation or synthesis, fatty acid biosynthesis, transport of metabolites, degradation of extracellular polysaccharides (preferably polymers of glucose, fructose, galactose, N-acetylglucosamine, glucosamine, mannose or chitobiose, or combinations thereof) or nitrogen metabolism.

In a preferred embodiment, the invention provides a method for controlling metabolism in a DasR-like protein comprising micro-organism, comprising regulating the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene whose product is part of a metabolic route, wherein the expression of said gene results in the presence of a corresponding product that directs the metabolism in said micro-organism to or from glycolysis or to or from the citric acid (TCA) cycle.

The inventors have identified a particularly surprising example of such metabolic control in Thermobifido fusca, where all the steps of glycolysis are controlled by a DasR-like protein (FIG. 17 and Table 9). Therefore, it is clear to the skilled person that modifying, for example, the activity of DasR will allow the control of glycolysis and TCA cycle in the microorganism.

Other DasR-regulated genes that are identified by the present inventors, include antibiotics or antibiotic clusters, (commercial interesting) enzymes and so on.

Therefore, the invention provides in yet another embodiment a method for obtaining expression of a product of interest in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding said product of interest or a DasR-binding site in operable linkage with a gene involved in the production of said product of interest.

Interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding said product of interest or a DasR-binding site in operable linkage with a gene involved in the production of said product of interest may be such that the binding is at least in part inhibited or it may be such that the binding is at least in part increased. With “at least in part” is meant that there is a detectable difference between the situation in which no compound is added and the situation in which said compound is added. In a preferred embodiment, the binding of a DasR-like protein to a DasR binding site is essentially completely inhibited or the binding is essentially irreversible.

Different compounds capable of influencing the binding between DasR protein and a DasR binding site in operable linkage with a gene that encodes a product of interest are already discussed above and include a genetic compound (i.e. a nucleic acid, optionally stably integrated in the genome of the corresponding micro-organism or existing as an episomal nucleic acid in said micro-organism) or a protein or a small hormone-like signalling molecule or for example a small (chemical) compound. In another preferred embodiment, said compound is an inducer of a DasR-regulated process. Such an inducer is especially useful in case of DasR-repressed expression. An example of such an inducer is glucosamine-6-phosphate or a functional equivalent or a functional fragment thereof. Preferably, N-acetylglucosamine (or a derivative or a multimer thereof) is provided to the medium of a micro-organism that is used for production of said product of interest.

A suitable compound is selected depending on the fact whether the expression of the gene corresponding to said product of interest is activated or repressed upon binding of a DasR-protein to a DasR binding site in operable linkage with said gene. In case expression is activated upon binding, it is desired to improve the binding of DasR protein to the binding site in operable linkage with the relevant gene, to enhance or improve or obtain expression. This is for example accomplished by adding DasR binding sites in operable linkage with the relevant gene, or by mutating the binding site (higher matrix score) to improve its interaction with DasR, or by adding DasR with improved binding capacity and so on. In case expression is repressed upon binding of DasR, it is desired to decrease binding of DasR to said binding site and thus to relieve said expression from repression. This is for example accomplished by deletion or mutation of the relevant DasR binding site.

In a preferred embodiment, said product of interest is a secondary metabolite. Examples of secondary metabolites are provided in Table 3 and include compounds such as clavulanic acid or butyrolactones.

Clavulanic acid is a very important inhibitor of beta-lactamase-type enzymes that are capable of inactivating penicillin and other beta-lactam-type antibiotics. Therefore, clavulanic acid is often administered to patients in combination with beta-lactam antibiotics such as penicillin when patients are expected to be infected with penicillin-resistant bacteria. Hence, a method according to the invention is also used to increase expression of clavulanic acid.

DasR also regulates genes involved in the biosynthesis of the hormone-like γ-butyrolactones, such as barB in S. virginiae (Table 3). These γ-butyrolactones are crucial signalling compounds that control antibiotic production and morphogenesis in actinomycetes (Horinouchi 2002; Beppu and Horinouchi, 1991; Horinouchi and Beppu, 1993; Horinouchi and Beppu, 1992).

For example, A-factor controls streptomycin production in S. griseus. Therefore, in another embodiment, the invention provides a method for indirectly regulating antibiotic production indirectly through the control of signalling molecules.

In yet another preferred embodiment, said product of interest is an antibiotic, an enzyme, a product from a cryptic gene cluster, an anti-tumor agent or an agricultural compound. Examples of enzymes are a cellulase, a pectinase, a lipase, an amylase, a chitinase, a mannanase, a xylanase, a protease, a peroxidase, a catalase, a laccase, or a sugar isomerase. Examples of antibiotics are the glycopeptide antibiotics, including vancomycin (produced by Streptomyces toyocaensis and Amylocatopsis orientalis), daptomycin (produced by Streptomyces roseosporus) and the teichoplanin-like compound A47934 (produced by Streptomyces toyocaensis), chloramphenicol (produced by Streptomyces venezuelae), streptomycin (produced by Streptomyces griseus), novobiocin (produced by Streptomyces spheroides), and also the well-studied model clusters for antibiotic production, namely actinorhodin and undecylprodigiosin of Streptomyces coelicolor. The latter two are controlled by the pathway-specific activator genes actII-ORF4 (#27 in Table 2) and redZ (#129 in Table 2), respectively. Also, the actinorhodin biosynthetic enzyme ActVA4 was strongly activated in the absence of DasR (FIG. 4). On the basis of our analysis of the known sequences related to antibiotic biosynthesis we surprisingly disclose that more than half of all known antibiotic biosynthesis clusters are regulated by DasR and hence intervention via DasR controlled expression provides a very useful means for modifying antibiotic production.

Moreover, the inventors also disclose that the actinorhodine biosynthese cluster is (indirectly) under control of DasR. As disclosed in FIG. 4B, a DasR null mutant secretes a considerable amount (approximately 95% of the total amount of secreted protein) of protein which is designated as SCO5074 (dehydratase). This dehydratase is part of the actinorhodine biosynthesis cluster and the dehydratase tailors the sectreted antibiotic. Hence, the invention further provides means and methods for controlling (preferably increasing) the amount of produced/secreted actinorhodine.

Both filamentous micro-organisms as well as low G+C gram-positive bacteria are examples of micro-organisms in which the methods according to the invention can be applied. However, using these micro-organisms in for example a method for obtaining expression of a product of interest can be accompanied by production of an undesired product. Some of these undesired products are now known to be regulated by DasR and the invention thus provides in yet another embodiment, a method for at least in part reducing production of an undesired product in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding said undesired product or a DasR-binding site in operable linkage with a gene involved in the production of undesired product.

Interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding said undesired product or a DasR-binding site in operable linkage with a gene involved in the production of said undesired product may be such that the binding is at least in part inhibited or it may be such that the binding is at least in part increased. With “at least in part” is meant that there is a detectable difference between the situation in which no compound is added and the situation in which said compound is added. In a preferred embodiment, the binding is essentially completely inhibited or is the binding is essentially irreversible.

Preferably, the binding of a DasR-like protein to a DasR-binding site is at least in part increased by providing said micro-organism with an increased amount of DasR-binding sites in operable linkage with a gene encoding said undesired product or in operable linkage with a gene involved in the production of said undesired product. In yet another preferred embodiment, the binding of a DasR-like protein to a DasR-binding site is at least in part decreased by removing at least one DasR binding site in said micro-organism that was originally in operable linkage with a gene encoding said undesired product or in operable linkage with a gene involved in the production of said undesired product.

As disclosed herein, the expression of some proteases are regulated by a DasR-like protein. Examples are provided in Table 2: #60 (metallopeptidase) or #109 (peptidase). In case a method of the invention is used to produce a proteinaceous product of interest, the expression of the mentioned peptidases can be influenced at the same time.

In a preferred embodiment, said undesired product is an undesired side product or an undesired shunt product.

It is clear to a skilled person that any of the methods according to the inventions can optionally be combined. For example a method for controlling metabolism combined with a method for at least in part reducing production of an undesired product. In a preferred embodiment, the invention provides a combination of a method for obtaining expression of a product of interest with a method for at least in part reducing production of an undesired product and hence, the invention provides a method for obtaining expression of a product of interest in a DasR-like protein comprising micro-organism as described above, further comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding an undesired product or a DasR-binding site in operable linkage with a gene involved in the production of an undesired product.

For example if the product of interest is encoded by a gene whose expression is repressed upon binding by DasR to a DasR binding site in front of said gene, expression of this product of interest is at least in part increased by providing an inducer. If at the same time an undesired product is expressed which presence for example interferes with the isolation/purification of said product of interest and if this undesired product is activated upon binding of a DasR protein to the relevant DasR binding site, this binding site is for example genetically modified to at least in part block binding of a DasR protein to said relevant DasR binding site. Based on the present invention it is clear to the skilled person that various situations of DasR regulation of the product of interest and the undesired product are possible which may be solved via any one of the herein described options.

Examples of genes whose expression is regulated by DasR (either activated or repressed) are outlined in Table 2. It is clear to the skilled person that the invention also provides methods based on the specific properties of the products of these genes. Moreover, we also have observed that the absence of DasR results in aberrant growth behaviour on certain growth media. The following part of the description provides examples of uses based on the results as presented in Table 2 as well as uses based on the observation with a DasR null mutant.

One example is the observation that the absence of DasR (see the experimental part related to the DasR null mutant) or the enhanced expression of DasR results in aberrant growth behaviour. For example, a DasR null mutant grown on glucose results in fragmented growth of a filamentous micro-organism. In liquid cultures the absence of DasR results in enhanced branching (FIG. 16), while expression of DasR using a multi-copy plasmid reduces branching. Moreover, the inventors have identified genes that are related to the cytoskeleton or peptidoglycan. Hence, the invention further provides a method for obtaining a filamentous micro-organism with altered fragmentation and/or branching characteristics during growth comprising altering the expression of a DasR-regulated gene. Most filamentous micro-organisms (for example streptomycetes) only sporulate on solid media, while growth in a liquid culture is restricted to the formation of a vegetative mycelium. This typically develops into an intricate network of hyphae, among others resulting in pellet formation, with only the most outwardly oriented sections showing high physiological activity, resulting in reduced growth rate and low yield of the desired product per unit of biomass. Furthermore, because of their filamentous morphology, high density fermentations of biotechnologically interesting streptomycetes often are highly viscous, resulting in a low biomass accumulation due to for instance aeration and mixing problems. From this perspective it is desirable that fragmentation of the mycelium in submerged cultures is stimulated, that branching of the mycelium is reduced, and that in general the viscosity of the culture is reduced.

Another example is based on the observation that some of the identified DasR regulated genes are genes involved in protein secretion and/or protein folding. Examples are secE (#102 in Table 2) and secY (#100 in Table 2), which are essential membrane components of the Sec secretion system. In fact, repeated measurements of total secreted protein in large cultures revealed that in the absence of an active copy of DasR total amount of secreted protein was reduced by 50%. Hence, the invention also provides a method for regulating protein secretion and/or protein folding in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene involved in protein secretion and/or protein folding.

Yet another example is based on the observation that some of the identified DasR regulated genes are genes involved in transport of metal ions, such as copper, iron or zinc. Relevant genes are for example a cutC-type copper homeostasis gene (#30 in Table 2) and the iron regulatory gene desR that controls iron uptake (#62 in Table 2). Hence, the invention also provides a method for regulating transport of a metal ion in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene involved in the transport of said metal ion.

Another example is based on the observation that many of the identified DasR regulated genes are genes involved in transport of sugars. Non-limiting examples are provided in Table I, including the PTS genes (nagE1, nagE2, malX2, ptsH, ptsI, crr; #1, 2, 3, 5), ABC transporters (e.g. #4, 8, 17, 22, 33, 53, 66, 111, and 122 in Table 2) and MsiK (#38) which is the universal ATPase for ABC transporters in streptomycetes and hence essential for their transporting activity. Of these examples, the PTS, MsiK and several of the ABC transporters have been experimentally validated. Hence, the invention also provides a method for regulating transport of a sugar in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene involved in the transport of a sugar. For example N-acetylglucosamine by the PTS^(NAG) and chitobiose by the ABC transporter NgcEFG (#33 in Table 2). The DasR-repressed MsiK is required for the activity of many ABC transporters, for example for transport of cellobiose (CebEFG), maltose (MalEFG) and xylobiose (BxlEFG).

Yet another example is based on the observation that the production of antibiotics in a dasR null mutant showed medium-dependent development: on some media development was enhanced, while on others it was completely abolished. Hence, the invention also provides a method for influencing antibiotic production in a (filamentous) micro-organism, comprising functionally impairing a dasR gene and further comprising selecting a sugar source suitable for a particular antibiotic. Examples of a suitable sugar source in combination with a particular antibiotic are provided in FIG. 11.

In any of the methods of the invention a DasR-like protein comprising micro-organism is a micro-organism that comprises a DasR-like protein or an organism that is capable of expressing said DasR-like protein from a nucleic acid encoding said protein. Moreover, said micro-organism may be an organism that is by nature capable of expressing said DasR-like protein or it may be a micro-organism which has been genetically modified to produce DasR-like protein. Moreover, it is also possible to provide a micro-organism that is already capable of expressing DasR-like protein with a further nucleic acid that enables production of more DasR-protein. Such an additional nucleic acid encoding a DasR-like protein may be identical to the already present nucleic acid encoding the endogenous DasR or it may be heterologous. In a preferred embodiment, a DasR-like protein comprising micro-organism is obtained by providing a micro-organism with a nucleic acid encoding a DasR-like protein. Even more preferably, said DasR-like protein is heterologous. A nucleic acid encoding a DasR-like protein may be provided to a micro-organism by any method known therefore. Moreover, said nucleic acid may be present in said micro-organism as an episomal element or integrated into the genome of said micro-organism.

Moreover, said gene of interest can be an endogenous gene as well as an exogenous gene and hence a method of the invention can further comprise providing a DasR-like comprising organism with a nucleic acid encoding a gene of interest.

A micro-organism used in any of the methods of the invention is preferably a bacterium from marine or soil origin. In another preferred embodiment, said micro-organism is a filamentous micro-organism. Examples of a filamentous micro-organism are an ascomycete, a basidomycete, or an actinomycete. Examples of a suitable actinomycete are a Streptomyces, a Nocardia, a Thermobifido, an Amycolatopsis, a Planobispora, a Streptoverticillium, a Rhodococcus, or a Corynebacterium.

In yet another embodiment of the invention, a micro-organism is a low G+C gram-positive bacterium, such a Bacillus, Lactobacillus, Lactococcus, Streptococcus, or Listeria. Surprisingly, the inventors disclose in the present invention the core gene cluster nagA-nagB-dasR (and the dre elements) is also widespread among low G+C Gram-positives, including Bacillus, Lactococcus, Listeria and Streptococcus. Not only the organization is conserved, but also the sequence of the dre sites, even though the G+C content of the DNA of Bacillus (43%) is around 30% lower than that of streptomycetes (72-73%). The dre sites of Bacillus subtilis and Bacillus halodurans are summarised in Table 5, those of Lactococcus lactic in Table 6, of Streptococcus species in Table 7, and of Listeria innocua and Listeria monocytogenes in Table 8.

Low G+C gram-positive bacteria are important in for example the production of many dairy products and moreover, these bacteria are often used as a probiotic.

It is clear from the Tables 4 to 7, that also in these bacteria processes such as sugar uptake, metabolism or bacteriocin production is influenced by any of the methods described herein.

Surprisingly, the invention also discloses the finding that a dasR mutant showed strongly enhanced production of antibiotic. Moreover, as identified in Table 3, multiple antibiotic (clusters) are under control of DasR. Also cryptic antibiotic (clusters) have been found to be under control of DasR (Table 3). The invention thus provides a method for regulating, obtaining or increasing the expression of an antibiotic in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding said antibiotic or a DasR-binding site in operable linkage with a gene involved in the production of said antibiotic. Preferably, such a method is used for obtaining or increasing the expression of an antibiotic. Even more preferably, said antibiotic is part of a cryptic antibiotic or a cryptic antibiotic cluster. In a preferred embodiment, said compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site is N-acetylglucosamine or a derivative or a multimer thereof.

In yet another embodiment, the invention provides an activated DasR-repressed cryptic gene (cluster), wherein said cryptic gene (cluster) is activated by influencing binding of a DasR-like protein to a DasR-binding site in operable linkage with said gene (cluster). Influencing the binding between said protein and said binding site may result in a decreased or an increased/improved binding. In a preferred embodiment, said gene (cluster) is an antibiotic gene (cluster).

In case the binding of DasR to the relevant binding site results in a repressed level of for example antibiotic production, production of said antibiotic is for example accomplished by mutating the DasR binding site in operable linkage with said antibiotic. The mutation is preferably such that a DasR protein is no longer capable of sufficiently binding to said binding site. In yet another embodiment an inducer such as N-acetylglucosamine (which is converted to glucosamine-6-P) is used to relieve repression.

The invention also provides a micro-organism that has been genetically engineered in such a way that the expression of at least one DasR regulated gene is modified.

One suitable example is a micro-organism comprising an activated cryptic cluster as described above, e.g. a micro-organism in which the DasR binding site in operable linkage with said cluster is mutated such that binding of a DasR-like protein is increased or decreased.

Another example is a micro-organism comprising a mutated binding site for a DasR-like protein. Said binding site is mutated such that binding of a DasR-like protein is increased or decreased. Alternatively, said binding site will be modified such that the genes are controlled by a different repressor protein (for example to specifically repress the genes whose expression is less desirable). A skilled person is aware that several DNA binding proteins exist that recognise only binding sites that do not naturally occur in a particular micro-organism. Examples of such proteins are TetR (recognizes the tetO operator) or Gal4 (recognizing the eukaryotic gal promoter).

Yet another example is a micro-organism comprising a mutant of a DasR-like protein. For example a mutant with improved binding capacity or with decreased binding capacity or a variant of DasR that is insensitive to induction by glucosamine-6-P (or similar compounds). Another example is an essentially non-functional mutant, for example a null mutant of a DasR-like protein. Yet another example is a DasR-like protein in which (part of) the effector binding domain has been mutated (for example deleted).

Another example is a micro-organism comprising increased expression of DasR. This is for example accomplished by providing a micro-organism with an (over) expression cassette for DasR. Such a cassette may comprise an endogenous or a heterologous dasR gene. If a micro-organism does already comprise the genetic information to encode DasR it is also an option to impair the autoregulation of DasR in said micro-organism.

Two other examples are a micro-organism which essentially grows vegetatively and which produces altered levels of a secondary metabolite or a micro-organism which essentially grows vegetatively and which produces a product from a cryptic gene cluster.

Preferably, any of the mentioned micro-organisms produces increased amounts of a product of interest (such as for example an enzyme or an antibiotic).

Even more preferably, a micro-organism according to the invention is a bacterium from marine or soil origin. In another preferred embodiment, said micro-organism is a filamentous micro-organism. Examples of a filamentous micro-organism are an ascomycete, a basidomycete, or an actinomycete. Examples of a suitable actinomycte are a Streptomyces, a Nocardia, a Thermobifido, an Amycolatopsis, a Planobispora, a Streptoverticillium, a Rhodococcus, or a Corynebacterium.

In yet another embodiment of the invention, a micro-organism is a low G+C gram-positive bacterium, such a Bacillus, Lactococcus, Streptococcus, or Listeria.

Although it is convenient to start any of the methods of the invention with a micro-organism that does already comprise a nucleic acid encoding a DasR-like protein as well as a nucleic acid in operable linkage with a DasR-binding site, it is also possible to use the methods of the invention with a micro-organism that does not comprise said nucleic acids. Hence, the invention further provides a method for regulating expression of a product of interest in a DasR-like protein comprising micro-organism, comprising providing a nucleic acid encoding said product of interest or a nucleic acid encoding a protein involved in the production of said product of interest with a binding site for a DasR-like protein and wherein said binding site is in operable linkage with said nucleic acid. In a preferred embodiment, said micro-organism is further provided with a nucleic acid encoding a DasR-like protein.

In yet another embodiment, the invention provides use a mutated DasR-binding site for regulating expression of a DasR-controlled gene or use of a mutated DasR-like protein for regulating expression of a DasR-controlled gene or use of DasR for regulating expression of a DasR-controlled gene or use of a DasR-inducer for regulating expression of a DasR-controlled gene.

Any of the herein-described methods may further comprise a purification step, for example purification of the product of a gene of interest or purification of a product of interest (such as an enzyme or an antibiotic). Moreover, a method according to the invention may be performed on a small-scale basis or on a large-scale basis.

In yet another embodiment, the invention provides a method for obtaining or improving production of a secondary metabolite in a micro-organism, comprising providing said micro-organism with N-acetylglucosamine or a derivative or a multimer thereof. Said secondary metabolite may be a known or unknown secondary metabolite and hence said method can be part of a screening method, for example a screening method for identification of a novel drug. In a preferred embodiment, said secondary metabolite is an antibiotic. In yet another preferred embodiment, the invention provides a method for awakening or enhancing expression from a cryptic (antibiotic) cluster in a micro-organism, comprising providing said organism with N-acetylglucosamine or a derivative or a multimer thereof. In another preferred embodiment, the used micro-organism comprises a DasR-like protein. In yet another preferred embodiment, the N-acetylglucosamine or a derivative or a multimer thereof is added to the growth medium of said micro-organism. In yet another preferred embodiment, said N-acetylglucosamine or a derivative or a multimer thereof is added to said growth medium at a concentration of at least or preferably above 10 mM.

In yet another preferred embodiment, the used micro-organism is a filamentous micro-organism, preferably an ascomycete, a basidomycete, or an actinomycete. A suitable example of an actinomycete is a Streptomyces, a Nocardia, a Thermobifido, an Amycolatopsis, a Planobispora, a Nonomuria, a Streptoverticillium, a Rhodococcus, or a Corynebacterium.

In yet another embodiment, the invention provides the use of N-acetylglucosamine or a derivative or a multimer thereof for awakening or enhancing a cryptic (for example antibiotic) cluster in a micro-organism.

The invention will be explained in more detail in the following description, which is not limiting the invention.

FIG. 1. Identification of the DasR-binding site and prediction of the DasR regulon. (A) DNase I footprint analysis of the DasR-binding site in the crr-ptsI promoter region (nt positions −202/+8 relative to the start of crr; SEQ ID NO:490). The crr-ptsI probe was incubated with DNase I (0.4 μg/ml) and increasing amounts of purified DasR (0, 10, 20, 40, 60, or 80 pmol of DasR in lanes 2-7, respectively). Additional controls: Lane 1, probe without DasR and without DNase I; lane 8, probe with DNase I and 350 pmol of non-specific protein (BSA). ACGT, DNA sequence lanes. The DNA sequence of the crr-ptsI probe is depicts the DasR-protected sequence (indicated with “FOOTPRINT”). The sequence that conforms to the DasR consensus (SEQ ID NO:491; below the target site) is highlighted in black, and the crr translational start codon in grey. (B) Pie chart showing categories and number of genes shown and/or predicted to be controlled by DasR (see Table 2).

FIG. 2. Effect of dasR on development and antibiotic production. (A) Antibiotic production by S. coelicolor M145 and its dasR mutant BAP29 on minimal medium agar plates with or without GlcNAc. The presence of GlcNAc is required for induction of the pigmented antibiotics actinorhodin and undecylprodigiosin in S. coelicolor M145, while the expression is constitutive in the dasR mutant. The latter is in line with our discovery that (i) ActV4A is enhanced in the mutant (FIG. 4) and (ii) the identification of a DasR-bound dre upstream of the pathway-specific activator genes actII-ORF4 for actinorhodin production and redZ for undecylprodigiosin production. Unexpectedly, the dasR mutant grown on minimal media with GlcNAc presents the expression of a yet unchartacterized green pigment. (B) Overexpression of DasR causes developmental arrest. Left, S. coelicolor M145 with control plasmid pUWL-KS; right, the same strain with a plasmid over-expressing dasR (grown for 4 days at 30° C.); note the almost complete lack of aerial hyphae (white) in the strain over-expressing DasR; (C) Scanning electron micrographs of aerial hyphae and spores of S. coelicolor M145 and the dasR mutant BAP29. Insert caption shows aberrant spores of the DasR mutant. Bars represent 5 μm. (D) Transmission electron micrographs of spores of M145 (left) and BAP29 (right). Arrows indicate the voids between the cell wall and the membrane. FD, full detachment; ID, incomplete detachment.

FIG. 3. Control of development via the PTS. (A) The PTS system is essential for correct development. Strains were grown for 5 days on SFM or R2YE agar plates. On SFM the crr mutant BAP2 produced a white aerial mycelium but failed to produce spores under these conditions, while deletion of ptsH (BAP1) or ptsI (BAP3) allowed the production of some grey-pigmented spores. Interestingly, on R2YE agar plates all three PTS mutants show vegetative arrest (so-called bald or bld phenotype). (B) PTS controls transcription of whiG. The figure shows a 1% agarose gel with amplification products from a semi-quantitative RT-PCR experiment. Total mRNA was prepared from exponentially grown mycelia of the wild-type and of PTS mutants BAP1 (ΔHPr), BAP2 (ΔIIA^(Crr)), and BAP3 (ΔEI). The figure shows that whiG-mRNA levels were diminished in the strains lacking the phophotransferases IIA^(Crr) and EI. Results were reproduced in triplicate from mycelia harvested at different time points within the exponential growth phase. Detection of 16S rRNA served as a standard reference control. (C) Control of posttranslational modification of WhiG by HPr. two-dimensional gel electrophoresis was performed on total protein extracts from S. coelicolor M145 (wt), and its pts mutant derivatives lacking the genes for HPr kinase, Enzyme IIA (Crr) or Enzyme I, respectively. A close-up of the area around the WhiG protein is shown. Expectedly, the strongly reduced transcripiton (3B) resulted in very low concentrations of the WhiG protein band in the mutants lacking Enzyme IIA and Enzyme I. Surprisingly, several bands with lower molecular mass than WhiG but with the same isoelectric point were absent from the HPr mutant, strongly suggesting involvement of HPr in the posttranslational modification of WhiG. The exact nature of the modification is unknown, but considering that the effect is only on mass and not on pI we believe that this is due to processing of a WhiG pre-protein.

FIG. 4. Identification of DasR targets by proteome analysis. (A) Close-ups of protein spots whose intensity depends on DasR (greater than 2-fold). Arrows highlight the protein spots identified by mass spectrometry. Arrows highlight the protein spots identified by mass spectrometry. 1, ThiL (SCO5399), acetoacetyl-CoA thiolase; 2, GdhA (SCO4683), glutamate dehydrogenase; 3, SCO5520, pyrroline-5-carboxylate dehydrogenase; 4, ActV-A4 (SCO5079), member of the actinorhodin biosynthesis pathway with unknown function; 5, MsiK (SCO4240), multiple sugar import protein; 6, GpsI (SCO5737), guanosine pentaphosphate synthetase; 7, SCO4366, phosphoserine aminotransferase; 8, NagA (SCO4284), N-acetylglucosamine-6-phosphate deacetylase; 9, GalT (SCO3138), galactose-1-phosphate uridylyltransferase. Proteins: ThiL (SCO5399), acetoacetyl-CoA thiolase; GdhA (SCO4683), glutamate dehydrogenase; SCO5520, Δ-1-pyrroline-5-carboxylate dehydrogenase; MsiK (SCO4240), multiple sugar import protein; GpsI (SCO5737), guanosine pentaphosphate synthetase; SCO4366, phosphoserine aminotransferase; NagA (SCO4284), N-acetylglucosamine-6-P deacetylase; GalT (SCO3138), galactose-1-P uridylyltransferase. (B) One-dimensional PAGE gel showing secreted proteins isolated from the spent medium of liquid-grown cultures of S. coelicolor M145 and its dasR mutant BAP29. M, molecular size marker (n kDa). Note that a single protein—identified as SCO5074 and also known as ActVI-ORF3—is strongly over-expressed in the dasR mutant BAP29.

FIG. 5. Schematic representation of N-acetylglucosamine-related enzymatic reactions and central position of glucosamine-6-P. DasR target identification is indicated: Arrows: enhanced (thick arrow) or inhibited (thin arrow) in the dasR mutant. Extracellular chitin is hydrolysed and once incorporated into the cell, converted to N-acetylglucosamine (GlcNAc), which is then phosphorylated by NagK to GlcNAc-6-P. NagA deacetylates GlcNAc-6-P to GlcN-6-P, which occupies a central position between nitrogen metabolism, the TCA cycle, peptidoglycan precursor synthesis, and glycolysis via NagB (converts GlcN-6-P to fructose-6-phosphate (Fru-6-P)). This schematic representation highlights the central position of glucosamine-6-P, the effector molecule of the DasR regulon. The large vertical arrow highlights the (dasR-controlled) flux from extracellular (poly-) sugars towards glycolysis and TCA cycleThe connected pathways can be retrieved from the KEGG database (http://www.genome.ad.jp/kegg/pathway.html).

FIG. 6. DasR activates chitin-related genes. (A) Comparison of the global chitinase, extra- and intracellular β-N-acetylglucosaminidase activities between BAP29 and M145 after 48 h of growth in minimal medium supplemented with various carbon sources. Activities are expressed in percentage (%) compared to the parental strain M145 under inducing conditions. (B) RT-PCR on chiI, chiF, and SCO6300.

FIG. 7. DasR controls the expression of principal polysaccharides degrading systems. Enzymatic activity was measured as the size of the clearing zone produced around the colonies due to specific degrading activity. Left, M145; right, dasR mutant BAP29. Top, assay of xylanase activity; middle, mannanase activity; bottom, α-amylase activity. CCR, carbon catabolite repression induced by glucose; SI, substrate induction induced by xylan (top), mannan (middle) and starch (bottom). Note that in the absence of dasR xylanase expression could not be induced, while mannanase and α-amylase activities were overexpressed. In CCR conditions all systems show an enhanced glucose repression.

FIG. 8. DasR represses genes the N-acetylglucosamine regulon (A) Effect of deletion of dasR on the uptake of GlcNAc. Left, uptake in glycerol-grown cultures; right, uptake in cultures grown in glycerol and inducer (GlcNAc); note that expression was constitutive in BAP29. (C) Western blot analysis of HPr (top) and IIA^(Crr) (bottom), showing that inducibility of these PTS proteins is controlled by DasR. (C) Transcriptional analysis of crr (for IIA^(Crr)), nagE2 (for IIC^(GlcNAc)), malX2 (for IIB^(GlcNAc)), nagB and chiF by semi-quantitative RT-PCR. Samples were collected before and 15, 30 and 60 min after the addition of GlcNAc to exponentially growing minimal medium glycerol cultures. 16S rRNA was used as control.

FIG. 9. cis/trans relationship between DasR and diverse target genes. Electromobility gel shift assays (EMSAs) are depicted that demonstrate binding of DasR to targets predicted in silico. Top: EMSAs were performed with 10 nM of fluorescent probe, without (upper plot) and with purified DasR (3 μM; lower plot) in the presence of 1000-fold excess of non-specific DNA. Bottom: EMSAs were conducted in a volume of 10 μl with 10 pmol of dre-containing DNA, a 1000-fold excess of non-specific DNA, and without (−) or with (+) 4 μg of purified DasR. DasR-dre complexes were resolved on 1% agarose gels in 1× TAE buffer. dre sites are indicated, where P_(xlnA) and P_(crp) served as negative controls and P_(crr) as the positive control.

FIG. 10. DppA activity measurements. Production of DppA (D-Ala-D-Ala aminopeptidase) by S. coelicolor M145 and BAP29 (ΔdasR) was measured as intracellular activity of DppA. Cultures were grown for 24 h in liquid MM cultures with various (combinations of) carbon sources, namely glycerol, glycerol+N-acetylglucosamine, chitin or glucose+chitin.

FIG. 11. Effect of sugars on antibiotic production in streptomyces coelicolor and effect of DasR. Act, actinorhodin (blue pigment); Red, undecylprodigiosin (red pigment); green, unknown new compound. Dev indicates developmental stage of the culture (bal, bald/vegetative phenotype; whi, white/aerial hyphae; spo, sporulation).

FIG. 12. DNA-binding by DasR is inhibited by glucosamine-6-P. The figure shows EMSAs of DasR binding to the nagB promoter. EMSAs were performed with 10 nM fluorescent probe, 3 μM purified DasR, and 500-fold excess of non-specific DNA. Plot 1 displays the control experiment on the nagB promoter (no DasR added); Plots 2-5, nagB promoter with DasR and 50, 100, 150, and 200 mM of GlcNAc (left panel, no effect) and GlcN-6-P (right panel, release of DasR from the dre site).

FIG. 13. Model for global control of carbon utilization by DasR. The model illustrates that abundant polysaccharidic carbon sources in the soil (cellulose, xylan, chitin, etc.) are degraded by extracellular hydrolases to the respective mono- and disaccharides. N-acetylglucosamine (GlcNAc) is transported by the PTS via sequential phosphoryl group transfer from PEP to enzyme I (EI, encoded by ptsI) to HPr (ptsH) to EIIA^(Crr) (crr), which in turn phosphorylates the EIIB protein of the GlcNAc-specific permease EIIBC. DasR functions as a repressor of all genes of the PTS^(GlcNAc). Other breakdown products are transported by ABC-permeases that are composed of two sugar-specific membrane proteins, a specific extracellular and a lipid-anchored sugar binding protein. Many of these ABC systems (more than 40 predicted) are assisted by the universal ATPase MsiK (multiple sugar import protein) that is regulated by DasR. The metabolic enzymes NagA and NagB (both genes controlled by DasR) convert GlcNAc-6-P to glucosamine-6-P (GlcN-6-P) and fructose-6-P. GlcN-6-P serves as an effector for DasR and thus provokes gene expression of pts genes and msiK. DasR-mediated control to nagB and to the dppA operon (D-Ala-D-Ala aminopeptidase) indicates its role in regulating cell wall synthesis. sh, sugar hydrolase gene; sr, gene for a specific regulator of an operon encoding an ABC permease and related extracellular sugar hydrolases. Yellow circles and red boxes on the fictive chromosome represent the DasR responsive elements and the sugar-specific regulator responsive elements, respectively.

FIG. 14. Alignment of DasR homologues from Gram-positive bacteria. (A) DasR homologues from actinomycetes. Homologues included in the pileup are DasR proteins from: Scoel, Streptonzyces coelicolor (SCO5231) (SEQ ID NO:492); Sclav, Streptonzyces clavuligerus (SEQ ID NO:493); Saver, Streptomyces avermitilis (SAV3023) (SEQ ID NO:495); Sgris, Streptonzyces griseus (BAB79296) (SEQ ID NO:496); Sscab, Streptomyces scabies (SEQ ID NO:494); S139, Streptomyces species 139 (AAN04228) (SEQ ID NO:497); Tfusc, Thermobifido fusca (AAZ54592) (SEQ ID NO:498). (B) Comparison of DasR-like proteins from high and low G+C Gram-positive bacteria. Interestingly, S. coelicolor DasR (Scoel; SEQ ID NO:492) is around 40% identical to Bacillus subtilis GntR-type protein CAB 15508 (Bsubt, SEQ ID NO:504). Additional proteins are from Nfarc, Nocardia farcinica (SEQ ID NO:499); Ceffi, Corynebacterium efficiens (BAC19131) (SEQ ID NO:501); Lmono, Listeria monocytogenes (AAT03756) (SEQ ID NO:502); and Sther, Streptococcus thermophilus (AAV62475) (SEQ ID NO:503). Proteins represented in (A) that are also represented in (B) include Sscab (SEQ ID NO:494); Saver (SEQ ID NO:495); sgris (SEQ ID NO:496); 5139 (SEQ ID NO:497); and Tfusc (SEQ ID NO:498). A second DasR homologue (designated DasR2; SEQ ID NO:500) was found in Streptomyces coelicolor (SCO0530) and is included in the pileup. The only known target site is located immediately upstream of the ABC transporter operon SCO531-532-533. (C) The similarity between the genes SCO5231-5235 and SCO530-534 strongly suggests a gene duplication event.

FIG. 15. Derived consensus sequences from Streptomyces, Bacillus, Lactococcus, Listeria and Streptococcus. The figures were prepared using the Logo software (Crooks et al., 2004). Large letters indicate high conservation, low letters indicate low conservation of the nucleotide position in all predicted DasR binding sites in the respective organisms. For these predictions we used the consensus DasR binding sequence from S. coelicolor to search for homologous sequences upstream of pts and nag genes in low G+C Gram-positive bacteria. The consensus sequences for the DasR binding sites in the respective organisms are, e.g.: Streptomyces (SEQ ID NO:1); Bacillus (SEQ ID NO:4); Lactococcus (SEQ ID NO:5); Listeria (SEQ ID NO:6); and Streptococcus (SEQ ID NO:7). The derived consensus sequences were then used to build a new matrix for putative DasR binding sites occurring in Bacillus, Lactococcus, Listeria and Streptococcus.

FIG. 16. Morphology and branching of S. coelicolor M145 and its dasR mutant in liquid-grown TSB cultures. While S. coelicolor M145 (top picture) shows typical occasional branching, deletion of the dasR gene results in strong increase in brancing (bottom picture). This suggests that modification of the expression level of dasR allows us to determine the branching frequency, which is important for the control of morphology and hence is a tool for improved growth behaviour in large scale fermentations. Bar=10 μm.

FIG. 17. Complete control of glycolysis and related pathways by DasR in Thermobifido fusca. Almost all steps in glycolysis and the connected pathways leading to oxaloacetate are predicted to be directly controlled by DasR. Database reference numbers for the respective genes are indicated. Note that every single step in glycolysis is predicted to be DasR-dependent.

FIG. 18. N-acetylglucosamine is transported by NagE2 (SCO2907). Mutants deleted for the transport genes nagE1/(SCO2906), nagE2 (SCO2907), or both, were plated on R2YE agar plates with or without N-acetylglucosamine (1% w/v). Other strains on the agar plates are: S.coelicolor M145 (parent of all mutants), the dasR mutant BAP29 and the pts mutants ptsH (BAP1), crr (BAP2), and ptsI (BAP3). For phenotypes of the pts mutants see also FIG. 3A). Excitingly, in the absence of nagE2 (or nagE2 and nagE1)addition of N-acetylglucosamine has no effect on development, while the nagE1mutant and the parental strain S. coelicolor M145 become arrested in the vegetative state. This proves that indeed nagE2 is the transporter of N-caetylglucosamine and is essential for import of the inducer molecule for the DasR control system.

FIG. 19. The nucleic acid and amino acid sequence of S. clavuligerus dasR/DasR. A) The dasR nucleic acid sequence (SEQ ID NO:505). B) The DasR amino acid sequence (SEQ ID NO:506).

FIG. 20. Alignment of protein sequences corresponding to the helix-turn-helix DNA binding motif of DasR obtained from various Streptomyces species. Sambo, S. ambofaciens (SEQ ID NO:517); Saver, S. avermitilis (SEQ ID NO:508); Scinn, S. cinnamoneus (SEQ ID NO:515); Scoel, S. coelicolor (SEQ ID NO:507); Scoll, S. collinus (SEQ ID NO:510); Sdias, S. diastatochromogenes (SEQ ID NO:514); Sgran, S. granaticolor (SEQ ID NO:512); Sgold, S. goldeniensis (SEQ ID NO:516); Sgris, S. griseus (SEQ ID NO:509); Slimo, S. limosus (SEQ ID NO:513); and Svene, S. venezuelae (SEQ ID NO:511). Amino acid numbering corresponds to aa sequence of S. coelicolor DasR (SCO5231). Symbols HTH above the sequence refer to the Helix-Turn-Helix DNA binding signature.

FIG. 21. DasR represses expression of Act and Red pathway-specific activators. (A) Electrophoretic mobility shift assays showing that DasR interacts with dre sites predicted upstream of actII-ORF4 and redZ. DNA probes encompassing dre sites found upstream of the crr-ptsI operon (encoding enzyme EIIA and enzyme EI of the PTS), actII-ORF4 and redZ were incubated with (+) or without (−) purified His-tagged DasR. The experimentally validated dre site upstream of the crr-ptsI operon (Rigali et al 2004) and the predicted cis-acting element of crp (Derouaux et al 2004) of S. coelicolor were used as positive and negative controls, respectively. (B). Transcriptional analysis of Act and Red pathway-specific activators by semi-quantitative RT-PCR. DasR directly represses transcriptional expression of actII-ORF4 and redZ. Samples were collected from S. coelicolor M145 and the dasR mutant grown on MM mannitol agar plates after 30 h (vegetative growth), 42 h (initiation of aerial growth), and 72 h (aerial growth and spores). v, vegetative mycelium; a, aerial hyphae; s, spores.

FIG. 22. N-acetylglucosamine-dependent signalling cascade of actinorhodin and undecylprodigiosin production in S. coelicolor. N-acetylglucosamine (GlcNAc) enters the cytoplasm and is subsequently phosphorylated via the GlcNAc-specific phosphoenolpyruvate-dependent phosphotransferase system, composed of intracellular general PTS proteins EI, HPr, and EIIA, and the GlcNAc-specific EIIB and EIIC components. N-acetylglucosamine-6-phosphate (GlcN-6P) is further deacetylated by NagA, the GlcN-6P deacetylase. The resulting glucosamine-6-phosphate (GlcN-6P) is a known allosteric effector of DasR able to inhibit its DNA-binding ability, resulting in loss of transcriptional repression of actII-ORF4 and redZ, which encode the pathway-specific transcriptional activators of the actinorhodin and undecylprodigiosin biosynthesis clusters, respectively. In support of the deduced antibiotic biosynthesis signalling cascade, GlcNAc induces Act and Red production in the S. coelicolor ΔredD (M510) and ΔactII-ORF4 (M511) mutants, respectively.

FIG. 23. Conservation of the GlcNAc-dependent antibiotic-inducing pathway amongst streptomycetes. Streptomycetes were grown on MM agar plates with 0.5% mannitol alone (left panel), or with added 1% GlcNAc (right panel), and an overlay was applied containing B. subtilis, allowing visualization of growth inhibition caused by antibiotic production. The tested streptomycetes were Streptomyces lividans 1326 (1326), Streptomyces hygroscopicus (hygro), Streptomyces collinus (colli), Streptomyces roseosporus (roseo), Streptomyces cinnamonensis (cinna), Streptomyces venezuelae (venez), Streptomyces clavuligerus (clavu), Streptomyces rimosus (rimos), Streptomyces griseus (grise), Streptomyces acrimycini (acrim), Streptomyces limosus (limos), and Streptomyces avermitilis (averm). See Materials and Methods section for exact nomenclature. The GlcNAc-triggering effect on antibiotic activity was most obvious for S. hygroscopicus, S. collinus, S. venezuelae, S. clavuligerus, S. rimosus, and S. griseus.

FIG. 24. DasR represses expression of the type I polyketide “cryptic” cluster. Transcriptional analysis of the cryptic type I polyketide cluster of S. coelicolor (SCO6273-5C06288) by semi-quantitative RT-PCR. Inactivation of dasR results in the transcriptional “awakening” of the cryptic pathway-specific activator gene, kasO (SCO6280), and subsequently enhanced transcription of SCO6273, encoding a putative type I polyketide synthase, during vegetative growth. Samples were collected from S. coelicolor M145 and the dasR mutant BAP29 grown on MM mannitol agar plates after 30 h (vegetative growth), 42 h (initiation of aerial growth), and 72 h (aerial growth and spores). v, vegetative mycelium; a, aerial hyphae; s, spores.

DESCRIPTION OF TABLES

Table 1 Experimentally validated DasR binding sites used to build the matrix for consensus sequence.

Table 2 Non-limiting list of genes controlled by a DasR binding site in Streptomyces coelicolor.

Table 3 List of putative binding sites for DasR relating to secondary metabolism (cut-off score 5). A. Antibiotics and metabolites of known function produced by actinomycetes B. Known and cryptic biosynthesis clusters of Streptomyces coelicolor.

Table 4 DasR target genes related to glutamate and glutamine metabolism.

Table 5 DasR binding sites in Bacillus species A. B. subtilis B. B. halodurans.

Table 6 DasR binding sites in Lactococcus lactis.

Table 7 DasR binding sites in Streptococcus species A. S. pneumoniae B. S. pyogenes C. S mutans D. S. agalactiae.

Table 8 DasR binding sites in Listeria species. A. Listeria innocua B. Listeria monocytogenes.

Table 9 DasR binding sites in Thermobifido fusca. Metabolic genes corresponding to FIG. 17 are highlighted.

DETAILED DESCRIPTION OF THE INVENTION Experimental Part Materials and Methods

Bacterial strains. E. coli DH5α, and BL21(DE3) were used for subcloning and DasR overexpression experiments. S. coelicolor M145, M510 (M145 ΔredD), M511 (M145 ΔactII-IV) and M512 (M145 ΔactII-IV ΔredD) (Floriano and Bibb, 1996) and Streptomzyces lividans 1326 were all obtained from the John Innes Centre strain collection, Streptomyces avermitilis NRRL 8165 (MA-4680), Streptonzyces hygroscopicus ATCC27438, Streptomyces limosus ATCC 19778, Streptomyces rinzosus ATCC 10970, Streptonzyces roseosporus ATCC 31568 and Streptonzyces venezuelae ATCC15439 were obtained from the ATCC strain collection and Streptomyces acrinzycini DSM 40540, Streptomyces cinnanzonensis DSM 40467, Streptomyces clavuligerus NRRL 3585, Streptomyces collinus DSM 40733 and Streptomyces griseus NRRL B2682 from the DSMZ strain collection. The dasR mutant BAP29 (ΔdasR::accC4) was created by replacing the coding region of the gene by the apramycin resistance gene cassette, using plasmid pWHM3, according to a routine procedure (Nothaft et al., 2003). The same strategy was used to craeate the knock-out mutants for ptsH (BAP1), crr (BAP2), ptsI (BAP3), nagE1 (BAP4), nagE2 (BAPS), and nagE1/E2 (BAP6). BAP1-3 were published previously (Nothaft et al., 2003). S. coelicolor strains were grown at 28° C. using tryptic soy broth without dextrose as complex medium (TSB, Difco) or minimal medium (van Wezel et al., 2005). E. coli cultures were grown in Luria-Bertani broth (LB) at 37° C. Phenotypic characterization of mutants was done on minimal medium agar plates with various carbon sources as indicated in the text (Kieser et al., 2000). Quantification of Act and Red was performed as described previously (Martinez-Costa et al., 1996).

DNase I footprinting. A 222-bp DNA fragment corresponding to the −202/+8 region relative to the start of S. coelicolor crr gene (SCO1390) was chosen for DNase I footprinting. The DNA fragment was amplified from chromosomal DNA by PCR. 50 fmoles of ³²P end-labelled probe were incubated with the relevant proteins (DasR-(His)₆ and/or BSA) and DNaseI (0.4 μg/ml) as described (Sambrook et al., 1989).

Computational prediction. Multiple alignments and position weight matrices were generated as described previously (Rigali et al., 2004) by the Target Explorer automated tool (http://trantor.bioc.columbia.edu/Target_Explorer/) (Sosinsky et al., 2003). The weight matrix was deposited as “DasR4”.

                                                               Score  matrix  (DasR 4) $\begin{matrix} A & 0.63 & {- 0.53} & {- 1.12} & {- 2.71} & {- 2.71} & {- 0.53} & {- 1.12} & {- 2.71} & 1.31 & {- 2.71} & 1.16 & {- 2.71} & {- 2.71} & 0.76 & {- 0.53} & {- 0.17} \\ C & {- 1.08} & {- 0.12} & {- 2.71} & {- 2.71} & {- 2.71} & {- 2.71} & 0.15 & {- 1.08} & {- 2.71} & {- 2.71} & {- 2.71} & 1.36 & 1.36 & {- 2.71} & 0.36 & {- 1.08} \\ G & {- 0.49} & {\; 0.93} & {- 1.08} & 1.13 & 1.36 & {- 2.71} & 0.81 & {- 2.71} & {- 2.71} & 1.36 & {- 1.08} & {- 2.71} & {- 2.71} & {- 2.71} & 0.54 & {- 2.71} \\ T & 0.10 & {- 2.71} & 1.16 & {- 0.17} & {- 2.71} & 1.16 & {- 1.12} & 1.24 & {- 2.71} & {- 2.71} & {- 1.12} & {- 2.71} & {- 2.71} & 0.49 & {- 1.12} & 0.98 \end{matrix}$

The minimum score obtained by a sequence scanned by matrix DasR4 is −38.55 and the maximum score is 17.25. According to the current experimental validations, a DasR-binding site could be defined as a sequence of 16 nucleotides that, when scanned by the DasR4 matrix, obtains a score comprised between higher than 6 (and up to 17.25). Illustratively, a truly and experimentally validated DasR-binding site with a score of only −2,97 has been found upstream of gdhA encoding a NADP-specific glutamate dehydrogenase.

Microscopy. Transmission electron microscopy (TEM) for the analysis of thin sections of hyphae and spores was performed with a Philips EM410 transmission electron microscope (Mahr et al., 2000). Phase contrast micrographs were produced using a Zeiss standard 25 phase-contrast microscope, and a 5 megapixel digital camera.

Sugar uptake. Uptake assays with 20 μM N-[¹⁴C]acetyl-D-glucosamine (6.2 mCi mmol⁻¹) into mycelia were performed as described (Nothaft et al., 2003).

Protein purification and Western blot. Purification of recombinant histidine-tagged DasR (Rigali et al., 2004) and Western blot analysis with antibodies raised against HPr and IIA^(Crr) have been described elsewhere (Nothaft et al., 2003).

RT-PCR. RNA was isolated from mycelium of S. coelicolor M145 and BAP29. Minimal medium cultures containing 50 mM glycerol were inoculated with spores and grown until OD₅₅₀ of 0.6 (exponential growth). N-acetylglucosamine was added at 0.5% and samples were taken after 0, 15, 30 and 60 minutes. RT-PCR analyses were conducted with the Superscript III one-step RT-PCR Kit (Invitrogen). RT-PCRs without reverse transcription were used as control for absence of residual DNA. For semi-quantitative analysis, samples were taken at three-cycle intervals between cycles 18 to 35 to compare non-saturated PCR product formation (van Wezel et al., 2005). Data were verified in three independent experiments.

Oligonucleotides used for the RT-PCR experiments described in FIG. 21B were:

For redZ (5′-CGACATGAAAGTGCAGGTGG-3′ (SEQ ID NO:518) and 5′-TC000CTFGGTCAGCAAAAGC-3′ (SEQ ID NO:519)), for actll-ORF4 (5′-GCTGCAGACGTACGTGTACCACAC-3′ (SEQ ID NO:520) and 5′-GCGTCGATACGGAGCTGCATTCC-3′ (SEQ ID NO:521)), for redD (5′-TCATGGGAGTGCGGAGAACGCG-3′ (SEQ ID NO:522) and 5′-CGCCCCACAGTTCGTCCACCAG-3′ (SEQ ID NO:523)), SC06273 (5′-CGGGGGCGAACTCGTCAAGGTC-3′ (SEQ ID NO:524) and 5′-GCCGAGATGTCGATGAGGACGCGG-3′ (SEQ ID NO:525)), for kasO (5′-GCGGGATGCTCAGTGAGCACGG-3′ (SEQ ID NO:526) and 5′-GACGAGGTCGGCGAGGACGGG-3′ (SEQ ID NO:527)) and for rpsI (5′-GAGACCACTCCCGAGCAGCCGC-3′ (SEQ ID NO:528) and 5′-GTAGCGGTTGTCCAGCTCGAGCA-3′(SEQ ID NO:529)).

Two-dimensional gel electrophoresis and protein spot identification. Mycelia of S. coelicolor M145 and BAP29 were grown in minimal medium with 50 mM glycerol, harvested at different time points within exponential phase, washed, resuspended in 20 mM HEPES, pH7.5 and 50 mM MgSO₄ and sonicated; cell debris was removed after centrifugation. DNA and RNA were eliminated by DNase and RNase treatment. The proteins extracts were dialysed twice at 4° C. against water, followed by addition of 6 M solid urea and 2M thiourea, TritonX-100 (2.5% (v/v)) IPG buffer (0.5% (v/v)), DTT (25 mM) and bromophenolblue. Membrane proteins were removed by ultracentrifugation for 1 h at 65.000 g. 1.5 mg of the cytoplasmic protein fraction was applied on 24-cm IPG strips (pH range 4-7) on an IPGPhor unit (Amersham/Pharmacia). The IPG strips were subjected to 12.5% polyacrylamide gels that were run on the Ettan DALT II system (Amersham/Pharmacia). The gels were stained with PhastGel Blue R and scanned. Proteome patterns were compared using two gel sets derived from independent experiments. Protein intensities were analysed by densitometric gray scale analysis with TINA software (Raytest). Protein spots were excised, subjected to in-gel digestion with trypsin and analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (Marvin-Guy et al., 2005).

Electromobility gel shift assay (EMSA). EMSAs were performed with fluorescent probes (10 nM) with an ALF express sequencer (Filee et al., 2001). Purified DasR (3 μM) and 1000-fold excess of non-specific DNA were used in the reaction mixture. Predicted cis-acting elements were taken from the promoter regions of actII-ORF4 (SCO5085; 5′-CACATTGAAATCTGTTGAGTAGGCCTGTTATTGTCGCCCC-3′ (SEQ ID NO:530)), and redZ (SCO5881; 5′-ACAAGATCTTCTTGAGGTGGAAACCACTTTCGTATCAGTCT-3′ (SEQ ID NO:531)). Known cis-acting elements upstream of crr (SCO1390; CCGTGAGGAGTGTGGTCTAGACCTCTAATCGGAACA-3′ (SEQ ID NO:532)), and crp (SCO3571; 5′-TGCGGCATCCTTGTGACAGATCACACTGTTTGGACT-3′ (SEQ ID NO:533)) were used as positive and negative controls, respectively. The 16 nt dre sites are underlined.

Enzymatic activities. Chitinase activity was determined as described previously (Zhang et al., 2002) using a colorimetric assay with carboxymethylchitin—Remazol Brilliant Violet 5R (Loewe Biochemica GmbH, Germany) as substrate. D-Ala-D-Ala aminopeptidase measurements were performed with D-Ala-paranitroanilide as substrate (Cheggour et al., 2000). BCA protein assay (Pierce) was used for determining protein concentrations.

Global antibiotic activity assays. Samples (1 μl) of diluted spore suspensions were spotted on minimal medium plates containing 0.5% mannitol with or without 1% GlcNAc and incubated three days at 28° C. For the bioassay, we inoculated 10 ml of molten soft nutrient agar (SNA) with 500 μl of a Bacillus subtilis overnight culture (0D₆₀₀ ˜1), and poured the mixture into square 12-cm-side Petri dishes. Plates were kept 2 h at 4° C. to solidify SNA and to allow diffusion of antibiotics produced, then incubated overnight at 30° C.

Experimental Part Results Prediction of the DasR Regulon.

To allow the S. coelicolor genome to be scrutinized for the occurrence of the DasR operator site, we performed DNaseI footprinting on the dre of the crr-ptsI operon, encoding the PTS enzyme IIA (IIA^(Crr)) and enzyme I (EI) (FIG. 1A). The protected sequence (TGTGGTCTAGACCTCT (SEQ ID NO:10)) corresponded to positions −130 to −115 relative to the start of crr, and had a 13 out of 16 by match to the derived DasR binding site consensus sequence (see below). This information was used to determine the dre sites of target genes that we had already validated (Table 1 and (Rigali et al., 2004)). Using this training set, we built a refined position weight matrix (“DasR4”; see materials and methods), resulting in an alignment matrix that was used to scan the complete S. coelicolor genome.

A genome scan revealed 160 dre sites for 131 transcription units, representing over 200 candidate genes. About 40% of the target genes are related to sugar or aminosugar metabolism, with N-acetylglucosamine (GlcNAc) as the central saccharidic component (FIG. 1B and Table 2). The relationship with GlcNAc also connects to the identification of cell wall-associated peptidases. The predicted DasR regulon further includes genes for nitrogen metabolism including genes related to glutamine/glutamate amino acid metabolism. The list of targets also includes 16 transcription factors (#27, 31, 46, 51, 56, 62, 67, 71, 77, 81, 84, 105, 119, 129, 130, and 131 in Table 2), suggesting an extensive level of indirect transcriptional control by DasR.

Phenotype of the dasR Mutant

A dasR null mutant (BAP29) was constructed by replacing almost the complete coding region (nt 14-635 out of 765) by the apramycin resistance cassette (aacC4), to study the role of DasR in vivo.

The dasR mutant of S. coelicolor showed medium-dependent development: on some media development was enhanced, while on others it was completely abolished. This is summarised in FIG. 11. This strongly suggests that the function of DasR depends on the carbon and nitrogen sources used.

Surprisingly, the dasR mutant showed strongly enhanced antibiotic production (FIG. 2A). Overproduction of DasR in strain M145(pFT241 dasR⁺) resulted in a reversed, non-sporulating bald (bld) phenotype (FIG. 2B). Closer inspection of the dasR mutant by cryo-scanning electron microscopy (cryo-SEM) showed that spores were almost completely absent in the dasR mutant, and aerial hyphae collapsed readily during sample preparation (FIG. 2C). Analysis at high resolution by Transmission EM of cross-sections from S. coelicolor M145 and BAP29 revealed that while M145 produced normal spores, the dasR mutant produced many spores (approximately 30%) with smaller or larger voids close to the spore wall (FIG. 2D; voids indicated by arrows), suggesting extensive detachment of the cytoplasmic membrane from the spore wall. Additionally, spore morphologies were significantly more heterogeneous; while wild-type spores typically have a size of 0.6 by 0.8 μm, the dasR mutant showed an unusually strong variation in spore sizes (0.5-1.4 μm in length, but with the same width of 0.8 μm). In addition, many mutant spores had a wall with a thickness similar to that of aerial hyphae, failing to create the typical thick spore wall. These observations connect well to our in silico predictions (Table 2) that dasR controls genes involved in the fate of peptidoglycan, including the genes for the metabolism of the precursor N-acetyglucosamine.

DasR and the Control of Development

How does DasR control the switch to development? Inclusion of pts genes in the dasR regulon allowed us to propose that at least one link is through the DasR-mediated control of the PTS. In a previous publication (Nothaft et al., 2003), we described normal but retarded development for the individual pts knock-out mutants, namely BAP1 (ΔptsH, the gene for HPr), BAP2 (Δcrr, the gene for IIA^(Crr)), and BAP3 (ΔptsI, the gene for enzyme I (EI)). More detailed phenotypic analysis of the PTS mutants revealed that while eventually all of the pts mutants were on some media able to produce spores, morphogenesis was significantly delayed on diverse complex and minimal media agar plates with the strongest differences when grown in the presence of mannitol and arabinose. Strains were grown for 5 days on SFM or R2YE agar plates. As shown in FIG. 3A, On SFM the crr mutant BAP2 produces a white aerial mycelium but failed to produce spores under these conditions, while deletion of ptsH (BAP1) or ptsI (BAP3) allowed the production of some grey-pigmented spores.

Interestingly, on R2YE agar plates all three PTS mutants show vegetative arrest (so-called bald or bld phenotype).

We recently discovered in a proteomics screen of these mutants that the expression patterns (in BAP2 and BAP3) or the modification patterns (in BAP1) of the WhiG protein, a key developmental σ factor for early aerial growth (Chater et al., 1989), strongly differed from those in the parental strain M145. To establish the expression of whiG, its transcription was analysed in all three mutants and in M145. Interestingly, whiG transcription was strongly reduced in the BAP2 and BAP3 mutants (FIG. 3B), providing a likely explanation for their failure to complete sporulation, since whiG mutants have a characteritic non-sporulating phenotype.

Hence, we propose that DasR acts as the nutrient sensor, and translates this through control of the PTS, which in turn controls whiG and—in view of the developmental arrest of the pts mutants—most likely at least one or more other early developmental genes. It might be noteworthy that the phosphotransferases EI, HPr, and IIA^(Crr) provide a perfect signalling system through reversible metabolite-dependent phosphorylation, which in other bacteria is used for diverse but always carbon-related responses (Brückner and Titgemeyer, 2002).

Proteome Analysis

To obtain an assessment of the effect of DasR, we compared the protein profiles of BAP29 and its parent M145. Protein extracts were prepared from mycelia grown in the presence of glycerol (a neutral carbon source) and analyzed by two-dimensional gel electrophoresis. About 4% of the protein spots on the Coomassie-Brilliant-Blue-stained gels were altered in intensity by more than two-fold and eight were identified by mass spectrometry (FIG. 4A). 11 of the most spectacular differences between M145 and BAP29 were analyzed, and of these we could positively identify nine proteins by mass spectrometry (FIG. 4A). Two of the proteins were predicted in our in silico screen, namely the multiple sugar import protein MsiK and NagA (N-acetylglucosamine-6-P deacetylase). Only nagA and msiK were included in Table 2 and contained predicted dre sites. The others could be related to central and to secondary metabolism (see below and FIG. 5). Binding of DasR to the dre in the msiK promoter region was demonstrated by EMSA (see below). According to a role of MsiK in the uptake of inducers of polysaccharides-degrading systems, the induction of these enzymatic arsenals (about a hundred of genes) should be also affected due to the dasR deletion (see Discussion).

The lack of a dre upstream of the other seven genes suggests indirect control of these genes by DasR. Visualising the metabolic pathways related to proteins identified by proteome analysis revealed that most gravitate around GlcNAc and glutamate metabolism, fitting well with the in silico and in vitro data presented above (FIG. 5).

DasR Controls Secondary Metabolism and Antibiotic Production in Actinomycetes

Interestingly, two of the targets identified in our proteomics screen, namely ActVA4 and GpsI, which are both up-regulated in the dasR mutant, are involved in the production of the antibiotic γ-actinorhodin (Bibb, 2005) which correlated well with the early an activated production of blue γ-actinorhodin in the dasR mutant (FIG. 2A). GpsI is the guanosine pentaphosphate (pppGpp) synthetase that synthesizes the ppGpp precursor. It has been established that the stringent factor ppGpp has a causal role in activating actII-ORF4 transcription (Hesketh et al., 2001). The high amount of GpsI in BAP29 suggests an increased pool of ppGpp precursors and therefore early and enhanced production of actinorhodin. The function of ActVA4 is unknown but the gene is included in the cluster responsible for actinorhodin production (20 genes) and depending on the transcriptional activator actII-ORF4 (Arias et al., 1999). As follows from Table 2, actII-ORF4 features among the predicted DasR target genes. Direct binding of purified DasR to the dre upstream of this gene is substantiated by our observation that DasR protein directly binds to a double-stranded oligonucleotide containing the dre element found in the actII-ORF4 promoter region (FIG. 9). This proves that indeed DasR controls actinorhodin production by binding to the pathway-specific activator gene for the synthesis of this exciting compound, suggesting that DasR plays a crucial role in the control of antibiotic production in actinomycetes.

Excitingly, comparison of the secreted proteins in abstracts of the dasR mutant and its parent S. coelicolor M145 by one-dimensional gel electrophoresis showed that one single protein was extraordinarily highly over-expressed in the dasR mutant (FIG. 4B). This protein was identified by Mass spectrometry as SCO5074. This protein was recently shown to be part of the actinorhodin biosynthesis cluster, and is a secreted dehydratase that is most likely responsible for tailoring of the secreted antibiotic (Hesketh & Chater 2003; Taguchi et al, 2000). The gene product most likely assists cyclisation-dehydration of the alcohol in the actinorhodin precursor to give the pyran ring, a reaction that can proceed spontaneously but far less efficiently without it. As described in Taguchi et al. (2000), the actVI-ORF3 disruption mutant produces less (about half as much) actinorhodin as the parent. This is in line with our observation that while the wild-type strain produced a blue pigment, the dasR mutant produced a purple/violet pigment, most likely a variant of actinorhodin due to the extreme over-expression of SCO5074.

Also highly interesting is that using the novel bioinformatics techniques described above, we identified dre sites upstream of many more genes involved in the regulation and/or production of antibiotics. These targets are summarised in Table 3.

Control of Enzyme Production

With 16 predicted genes, chitin-related (chi) genes constitute a large subset of potential DasR targets, including chitinases, chitin binding proteins, extracellular β-N-acetylglucosaminidases (convert chito-oligosaccharides into GlcNAc and chitobiose), and intracellular β-N-acetylglucosaminidases (hydrolyse chitobiose to GlcNAc). To substantiate this, we determined the overall chitinolytic activity of BAP29 and M145 grown under inducing (chitin) or repressing (glycerol, glycerol plus GlcNAc, and glucose plus chitin) conditions. As depicted in FIG. 6A, we observed a strongly reduced chitinolytic activity in BAP29, when cells were grown on chitin in the presence or absence of glucose. Similar observations were made when total β-N-acetylglucosaminidase activities were assayed FIG. 6A).

Seven examples were selected to validate the predicted cis-trans relationship between DasR and chi genes, for the chitinolytic system that is required for the utilization of chitin, a polymer of N-acetylglucosamine that is the one but most abundant carbon source on earth. Positive DNA-DasR interactions were observed for all tested promoters (FIG. 9), although some had low binding efficiency. The transcription of three chitinase genes was monitored by RT-PCR on RNA isolated from cultures grown under conditions inducing (glycerol and GlcNAc) or not inducing (glycerol) the uptake of GlcNAc (FIG. 6B). The genes analysed were chiI (SCO1444), chiF (SCO7263), and SCO6300, encoding a putative secreted β-N-acetylglucosaminidase. As deduced from the global chitinolytic activity, a basal expression was observed for all three genes in cultures grown solely on glycerol. For chiI and SCO6300, there was no significant difference in transcript levels between M145 and BAP29. Excitingly, chiF transcription was fully dependent on DasR: while we failed to detect any transcript in RNA preparation from BAP29, there was strong chiF transcription in the RNA samples of the wild type. These data suggest that DasR positively controls the chitinolytic system, in contrast to its repressing function towards genes for GlcNAc transport and its subsequent intracellular catabolism (see below).

Other extracellular enzymes are also controlled by DasR (FIG. 7). Indeed, we found that besides the chitinolytic system also the activity of mannanases, α-amylases, xylanases depend on DasR (FIG. 7). All of these polysaccharide-degrading systems were in fact affected in both substrate induction and glucose control by dasR, underlining its crucial position in the control mechanisms for enzyme secretion.

DasR-Mediated Control of Sugar Transport

Since many of the predicted DasR targets were involved in the fate of carbon sources (Table 2), we analyzed the effects of the dasR mutation on sugar import. Transport assays revealed that PTS-mediated internalization of GlcNAc had become constitutive in BAP29, while in the parent M145 uptake was induced by GlcNAc (FIG. 8A). This correlated to constitutive protein levels of the universal PTS phosphotransferases HPr and IIA^(Crr) (FIG. 8B), and was supported by RT-PCR of the respective genes (malX2, nagE2, crr-ptsI and ptsH; FIG. 8C) that encode the PTS permease complex (IIB^(GlcNAc), IIC^(GlcNAc), IIA^(Crr), EI, HPr).

As shown above, in silico prediction and proteome analysis identified msiK (SCO4240), encoding the universal ATPase MsiK, as a target for DasR (FIG. 4). DNA binding experiments showed that DasR directly binds to the dre present in the msiK promoter region (FIG. 9), showing that DasR is involved in the regulation of MsiK-dependent ABC-type (ATP-binding cassette) transporters, which include those for uptake of cellobiose, trehalose, maltose, xylobiose, chitobiose, and probably another further 20 to 30 carbohydrates (Bertram et al., 2004). An obvious consequence of DasR-dependent control of MsiK is that DasR indirectly controls the availability of sugar operon inducers, thus affecting the expression of all extracellular sugar hydrolases. This corresponds well to our discovery that besides the chitinolytic system also the expression of mannanases, α-amylases, xylanases depends on DasR (FIG. 7).

DasR-Dependent Peptidoglycan-Associated Proteins

The observed cell-wall anomalies in the dasR mutant (FIG. 2D) are at least in part explained by the finding that several genes encoding peptidoglycan-associated peptidases are included in the list of potential DasR targets (Table 2). In fact, a site has been predicted 71 bp upstream a five-membered dppA operon (SCO6486-6490). dppA itself encodes a putative binuclear zinc-dependent, D-specific aminopeptidase (pfam 04951), 30% identical and 50% similar to DppA of Bacillus subtilis (DppA^(Bsu)) (Cheggour et al., 2000); DppA^(Bsu) is only active on D-Ala-D-Ala and D-Ala-Gly-Gly substrates. The physiological role of DppA^(Bsu), is probably an adaptation to nutrient deficiency by hydrolysing the D-Ala-D-Ala dipeptide required in peptidoglycan biosynthesis (Cheggour et al., 2000). An other orf of the dppA operon (SCO6489) is also involved in peptidoglycan precursors or peptidoglycan degradation products catabolism. The predicted gene product of SCO6489 is 32% identical and 47% similar to LdcA (L,D-carboxypeptidase A) from E. coli that hydrolyses the peptide bond between the di-basic amino acid and the C-terminal D-alanine in the tetrapeptide moiety in peptidoglycan (Templin et al., 1999). The inactivation of ldcA in E. coli results in a dramatic decrease in the overall cross-linkage of peptidoglycan.

To assess whether DasR controls the expression of the dppA operon, we performed DNA binding studies with purified His-tagged DasR and a fragment corresponding to 193 by upstream of dppA. Analysis using EMSAs established a weak but significant interaction of DasR with the dppA promoter (FIG. 9). To further substantiate a regulatory role for DasR on the expression of dppA, the intracellular D-Ala-D-Ala (SEQ ID NO:) aminopeptidase activity was measured in mutant BAP29 and compared to the parental strain S. coelicolor M145. Both strains were grown for 24 hours in MM supplemented with various carbon sources (FIG. 10). We failed to detect substantial variation in the total D-ala-D-Ala (SEQ ID NO:) aminopeptidase activity between M145 grown in chitin. However, in glucose+chitin we measured an average 85% of loss of activity in mutant BAP29 compared to M145. In glycerol and glycerol+GlcNAc the dasR mutant had about 70% and 33% increased activity, respectively, thus revealing an opposite effect. These experiments show that DasR controls dppA activity according to the culture conditions and therefore modulates the D-Ala-D-Ala pool required for peptidoglycan precursors biosynthesis.

DasR and Central Metabolism

Considering the large number of N-acetylglucosamine-related genes in the list of predicted dre sites, we investigated the impact of DasR on the regulation of the nag metabolic genes. EMSAs were conducted using purified DasR protein and DNA fragments encompassing the predicted dre sites for nagB (Glucosamine-6-P isomerase) and the nagKA operon (GlcNAc kinase, and GlcNAc-6-P deacetylase). In both cases a DasR-dre complex could be demonstrated (FIG. 9). This is consistent with our proteome analysis on NagA (FIG. 4), and with RT-PCR analysis of nagB, which is constitutively expressed in the dasR mutant (FIG. 8C). The DasR regulon further focuses on the fate of N-acetylglucosamine through control of genes for nitrogen metabolism, including aminosugar and glutamine/glutamate metabolism. Our proteome analysis revealed glutamate dehydrogenase (GdhA; completely dependent on DasR) and phosphoserine aminotransferase (SCO4366, repressed) as targets, which catalyse opposite reactions (Altermann and Klaenhammer, 2005) (FIGS. 4 & 5). Acetate that is liberated from N-acetylglucosamine by NagA is converted by acyl-CoA synthetase (strong dre site upstream of SCO3563 and confirmed by EMSA) to acetyl-CoA, the precursor of the TCA cycle. Acetyl-CoA is alternatively converted by a thiolase (ThiL; a target detected by proteomics, FIG. 4) to acetoacetyl-CoA to enter fatty acid metabolism. This may well extend to an unusual type of control at the translational level, as the last two genes in the operon containing all major tRNAs for Gln (anticodon CUG) and Glu (anticodon CUC)—in the order tRNA^(Gln)-tRNA^(Glu)-tRNA^(Glu)-tRNA^(Gln)-tRNA^(Glu)— are predicted to be regulated by DasR, while the first three are not, suggesting fine-tuning of tRNA availability by DasR. Supporting evidence for such control at the level at tRNA abundance comes from the presence of a predicted dre site upstream of Glu-tRNA^(Gln) amidotransferase (Table 2).

Hence, a picture emerges of a hyper-controlled core network of the DasR regulon, crucial for the cell's energy balance and revolving around the triangle GlcNAc-Gln/Glu-Acetyl-CoA, with almost complete control of all metabolic steps involved. Thus, DasR plays a particularly prominent role in the control of central metabolism and is a very attractive target for metabolic engineering. All targets relating to N-acetylglucosamine and glutamate metabolism are highlighted in Table 4.

Amazingly, our analysis of the Thermobifido furca genome showed that in fact every single step of glycolysis is controlled by DasR, with highly reliable dre sites located upstream of the respective enzyme-encoding genes (Table 9 and FIG. 17). The implications of this are truly daunting, as it means that in this industrially relevant actinomycete the flux through glycolysis can be easily controlled by the enhanced or reduced expression (or inactivation) of DasR.

Glucosamine-6-P is an Effector of DasR

A pivotal question is what is the effector molecule that modulates DasR? As shown in here, many of the targets for DasR relate to the generation (chitinolytic system), transport (PTS^(GlcNAc)), and metabolism (glycolysis via fructose 6-P) of N-acetylglucosamine. We therefore looked for the inducer among the intermediate molecules that gravitate around aminosugar metabolism. A binding interference experiment was set up where the ability of compounds to interfere with binding of dasR to the nagB and crr promoters was tested. These compounds were: N-acetylglucosamine, N-acetylglucosamine-6-P, glucosamine-6-P, glutamate, glutamine, acetyl-CoA, and fructose 6-P. These binding interference experiments identified glucosamine-6-P as the inducer/effector molecule, as it was the only of the compounds tested that prevented the formation of a complex of DasR with the nagB (FIG. 12) and crr promoter regions (not shown). The finding that glucosamine-6-P serves as an effector of DasR is explained by its central position at the metabolic crossroads between (GlcNAc)_(n) extracellular degradation, N-acetylglucosamine transport and intracellular metabolism, lipid and nitrogen metabolism, glycolysis, and peptidoglycan synthesis (FIG. 13).

N-Acetylglucosamine is Transported by the NagE2 Transporter

Two possible transporters for N-acetylglucosamine were identified on the S. coelicolor genome, namely the adjacent genes nagE1 (SCO2906) and nagE2 (SCO2907). Mutants were created for both genes by replacing the entire gene by the apramycin resistance cassette aacC4. A double mutant was also produced (BAP6). The method used to do this was by using pWHM3, as described previously (Nothaft et al., 2003). Mutants deleted for the transport genes nagE1 (BAP4), nagE2 (BAPS), or both (BAP6), were plated on R2YE agar plates with or without N-acetylglucosamine (1% w/v). Other strains on the agar plates are: S.coelicolor M145 (parent of all mutants), the dasR mutant BAP29 and the pts mutants ptsH (BAP1), crr (BAP2), and ptsI (BAP3). For phenotypes of the pts mutants see also FIG. 3A). Excitingly, in the absence of nagE2 (or nagE2 and nagE1) addition of N-acetylglucosamine has no effect on development, while the nagE1 mutant and the parental strain S. coelicolor M145 become arrested in the vegetative state. This proves that indeed nagE2 is the transporter of N-acetylglucosamine and is essential for import of the inducer molecule for the DasR control system.

Clearly, influencing the activity of NagE2 (positively or negatively) will have a strong effect on the amount of inducer molecules introduced into the Streptomyces cell, and therefore will strongly effect the DasR regulatory system.

The Full Scale of the DasR Regulon

While we describe here around 200 targets (Table 2), the true number is without doubt much larger; for example, we used a highly restrictive dre position weight matrix to avoid false positives, but we have evidence that by doing so many true dre sites have been obscured. Additionally, we identified at least eight transcription factor genes in the list of predicted DasR targets. DasR controls many sensing/transport elements and the expression of Glu- and Gln-tRNAs. This suggests that DasR may be receptive to diverse environmental changes and governs many other regulons, and most likely at both the transcriptional and at the translational level. This multi-level control by DasR is summarized in FIG. 13.

Besides the absolute size of the regulon, a prerequisite for a global-acting regulator is further that it should act in concert with single-acting transcription factors (Moreno et al., 2001). Indeed, DasR controls chi-related genes, which are also regulated by the ChiS/ChiR two-component system (Kormanec et al., 2000) and by a third unknown DNA-binding protein identified recently (Fujii et al., 2005), suggesting a multi-partner control of the chitinolytic system. Another example arises from our studies on the regulation of the PTS, where we observed that besides DasR also SCO6008, encoding a ROK-family regulator (Titgemeyer et al., 1994), is required for activation of pts genes.

The Wide-Spread DasR Regulon is a Target for Novel Screening Procedures

Excitingly, the DasR regulatory network is highly conserved in S. avermitilis and S. scabies, with more than 75% of the dre sites predicted in S. coelicolor also found upstream of the orthologous genes in S. avermitilis, providing a strong phylogenetic argument for the presented predictions. The strong conservation of the dasR regulon in other actinomycetes also suggests that DasR may control many genes for natural products and enzymes in this class of bacteria. The conservation of the DasR regulon is underlined by the high conservation of DasR proteins (FIG. 14).

Considering the predicted control of clavulanic acid production in S. clavuligerus (Table 3), we cloned the dasR gene by PCR using oligonucleotides matching the −50/−30 and +900/+920 regions of S. coelicolor dasR, with nt positions relative to the start of the gene. The clone was sequenced, and the predicted gene product differed in a single amino acid position, namely an Asn55 in S. clavuligerus DasR and Asp55 in S. coelicolor DasR. On this basis it is obvious that the DasR binding site in S. clavuligerus is highly similar to that in S. coelicolor. The corresponding nucleic acid and amino acid sequence are disclosed in FIG. 19.

Surprisingly, we recently discovered that the core gene cluster nagA-nagB-dasR (and the dre elements) is also widespread among low G+C Gram-positives, including Bacillus, Lactococcus, Listeria and Streptococcus. Not only the organization is conserved, but also also the sequence of the dre sites, even though the G+C content of the DNA of Bacillus (43%) is around 30% lower than that of streptomycetes (72-73%). The dre sites of Bacillus subtilis and Bacillus halodurans are summarised in Table 5, those of Streptococcus species in Table 7, of Lactococcus lactis in Table 6, and of Listeria innocua and Listeria monocytogenes in Table 8. The derived consensus sequences for DasR binding sites in these species are summarised in cartoons in FIG. 15.

From this we conclude that the DasR core regulon is a very important concept, as its presence in such divergent micro-organisms means that the DasR control system has survived at least half a billion years of evolution. Finding a tool to manipulate the activity of DasR is therefore very important, as it will allow controlling the expression of many industrially and medically relevant compounds (enzymes, antibiotics, anti-tumor agents, agricultural compounds, and other secondary metabolites) from the outside rather than by genetic engineering. This is for example a prerequisite for setting up novel screening strategies, as individual strain manipulation is not an option. Addition of inducer (notably N-acetylglucosamine and derivatives) will trigger or at least enhance the expression of a wide range of natural products, allowing more ready screening. An obvious example is the control of cryptic clusters, which are silenced and therefore cannot be identified by activity-based screening assays. We show that antibiotic biosynthesis clusters are activated by the removal or reduced activity of DasR, and we anticipate that addition of inducer will relieve these clusters and thus boost the potential of novel screening procedures.

Detailed Analysis of Effect of DasR on Act and Red Production

As shown above, on media that do allow development (e.g. mannitol-containing solid media) the dasR mutant showed enhanced production of the pigmented antibiotics actinorhodin (Act) and undecylprodigiosin (Red). The relative increase in antibiotic production in the dasR mutant was quantified by determining Act and Red concentrations in the spent agar of solid-grown cultures (MM without any added carbon source). Under these conditions S. coelicolor grows solely on agar, enabled by induction of the DagA agarase (Buttner et al., 1987). Spectroscopic measurements showed that Act and Red production were consistently enhanced in BAP29 by factors of 3.2 (±0.2) and 3.9 (±0.3), respectively (averages of three independent experiments). As shown in FIG. 4, in further support of enhanced Act production by the dasR mutant, preliminary proteome analysis of extracellular fractions of M145 and BAP29 identified two proteins encoded by genes in the act cluster that were strongly up-regulated in the dasR mutant, namely ActVI-ORF3, encoded by SCO5074, a secreted protein involved in stereospecific pyran ring formation of actinorhodin (Hesketh and Chater, 2003; Ichinose et al., 1999), and ActVA-ORF4, the product of SCO5079 (Caballero et al., 1991)), a conserved hypothetical cytoplasmic protein.

We observed putative dre sites upstream of actII-ORF4 and redZ encoding transcriptional activators of the act and red gene clusters, respectively (for dre sites see Table 2). The dre site upstream of actII-ORF4 (nt positions −59/−44 relative to the translational start site) lies precisely between the canonical −35 and −10 sequences of the promoter, a position strongly suggesting that DasR should function as a transcriptional repressor. The dre site upstream of redZ (nt positions −201/186 relative to the translational start site) lies around 50 bp upstream of the -35 sequence of the redZ promoter.

Electrophoretic mobility gel shift assays (EMSAs) with purified His₆-tagged DasR and double-stranded oligonucleotide probes showed direct binding to the predicted dre sites of redZ and actII-ORF4 and to the positive control (dre site of crr-ptsI), while DasR did not bind to the cis-acting element of crp, which lacks similarity to the dre element and was therefore used as the negative control (FIG. 21A). No free template was found when DasR was bound to the crr fragment, while over 50% of the redZ probe was bound, and only around 10% of the probe containing the actII-ORF4 dre site. Hence, we established direct binding of the DasR protein to the predicted dre sites, with binding efficiencies corresponding to their ‘statistical strength’.

The role of DasR in the control of actII-ORF4, redZ and redD was further assessed by semi-quantitative RT-PCR on RNA samples collected from the parental strain (M145) and the dasR mutant (BAP29) grown on MM mannitol agar plates for 30 h (vegetative growth), 42 h (initiation of aerial growth), and 72 h (aerial growth and spores). RT-PCR analysis revealed strongly enhanced transcription of actII-ORF4 at all time points in the dasR mutant and a discrete but significantly enhanced transcription of redZ (FIG. 21B). Apparently, not only does the degree of DasR-dependent transcriptional repression relate to the strength of the DNA-protein interactions, but the relative positioning of the dre site with respect to the promoter consensus sequence is also an important factor. It was shown previously that enhanced expression of redZ strongly induces redD transcription, even by-passing the block in antibiotic production in bldA mutants (Guthrie et al., 1998). Indeed, the enhanced expression of redZ was reflected in a clearly enhanced expression of the red pathway-specific activator gene redD. In conclusion, the known activator genes of the red cluster are negatively controlled by DasR, explaining the enhanced production of Red in a dasR mutant. For the act cluster, DasR competes with the transcriptional activator AtrA for binding to the promoter region of actII-ORF4, and inactivation of DasR—either through modulation of its in vivo activity or through gene inactivation—then results in enhanced Act production.

More Evidence that N-Acetylglucosamine Targets DasR to Unlock Antibiotic Production

A signalling cascade from initial detection of the nutritional status of the environment to the onset of physiological and chemical differentiation should contain at least the following steps: (1) availability and sensing of an extracellular signal; (2) transport of ‘signalling nutrients’ into the cell; (3) their intracellular modification into an inducer molecule; (4) its binding to a global regulator, which is the checkpoint for (5) signalling the information to pathway-specific activators and (6) the switch to development and antibiotic production. Our experiments suggest that the GlcNAc sensory cascade controlled by DasR is a global system that triggers antibiotic production in direct response to nutrients (FIG. 22). The steps are: (1) sensing of GlcNAc; (2) transport via the PTS^(GlcNAC); (3) conversion by NagA to glucosamine-6-P; (4) binding of the signalling molecule to DasR, thus inhibiting its repressing activity on actII-ORF4 and redZ and activating the pathways for biosynthesis of actinorhodin and undecylprodigiosin.

Arguing from the regulatory pathway deduced from the newly extended characterization of the DasR regulon (FIG. 22), we anticipated that DasR-dependent transport and phosphorylation of GlcNAc via the PTS could be a decisive signal to trigger actinorhodin and undecylprodigiosin biosynthesis in S. coelicolor. This hypothesis was tested by plating S. coelicolor M510 (ΔredD), M511 (ΔactII-ORF4) and M512 (ΔredD, ΔactII-ORF4) on MM agar plates with or without GlcNAc. As a consequence of the deletion of the respective pathway-specific activators, S. coelicolor M510 cannot produce the red-pigmented undecylprodigiosin and M511 fails to produce the blue-pigmented actinorhodin, while M512 produces neither antibiotic. These strains allowed us to specifically monitor each of the antibiotics, which is a necessary control because their pigmentation is pH-dependent, and biosynthetic derivatives show varying colours (Bystrykh et al., 1996; Ichinose et al., 1999). Neither strain produced significant amounts of actinorhodin or undecylprodigiosin when grown for five days on MM with agar as the sole carbon source. In the presence of GlcNAc (10 mM), production of Act or Red was induced in S. coelicolor M510 and M511, respectively (FIG. 22). No pigmented antibiotic was observed for M512, as expected (not shown). Thus, under starvation conditions production of Act and Red is induced both in response to increased levels of GlcNAc and by the absence of DasR.

Application of GlcNAc for Drug Discovery

Is the GlcNAc-mediated control of antibiotic production a more widespread phenomenon in streptomycetes? To assess this, we evaluated the effect of GlcNAc on total antimicrobial activity (bactericidal and bacteristatic) of several streptomycetes, using Bacillus subtilis as the indicator strain. The tested strains were spotted on minimal medium containing mannitol (0.5%) with or without GlcNAc (1%). Excitingly, growth inhibition zones indicative of antibiotic production were much larger for Streptomyces clavuligerus (a producer of cephamycin), Streptomyces collinus (produces kirromycin), Streptomyces griseus (streptomycin producer), Streptomyces hygroscopicus (produces hygromycin), Streptomyces rimosus (produces oxytetracycline), and Streptomyces venezuelae (chloramphenicol, methymycin) (FIG. 23). N-acetylglucosamine did not seem to affect antibiotic activity against B. subtilis in Streptomyces acrimycini, Streptomyces avermitilis Streptomyces cinnamonensis, Streptomyces limosus, and Streptomyces lividans. Interestingly, we observed a repressing effect in Streptomyces roseosporus. These results suggest that the relief of antibiotic production by GlcNAc (and through DasR) is a common control mechanism in streptomycetes.

In order to assess if DasR plays a role in silencing cryptic secondary metabolite clusters in streptomycetes, we analysed the expression level of a putative antibiotic biosynthetic cluster for a hypothetical type I polyketide (SCO6273-6288), the only cryptic cluster studied so far. Induction of this biosynthetic pathway depends on a pathway-specific activator, KasO (SCO6280), which is in turn repressed by the γ-butyrolactone (SCB1) binding protein ScbR (Takano et al., 2005). Repression of kasO is relieved by production of SCB1. To test the possible “awakening” of this cluster by the absence of dasR we performed semi-quantitative RT-PCR on RNA samples collected from the parental strain (M145) and the dasR mutant (BAP29) grown on MM mannitol agar plates for 30 h (vegetative growth), 42 h (initiation of aerial growth), and 72 h (aerial growth and spores). Excitingly, kasO transcripts were detected in the 30-h and 72-h RNA samples of BAP29, but were not seen in M145 in any of the samples (FIG. 24). Most likely as a result of the induction of kasO, transcription of SCO6273, the last ORF of the biosynthetic cluster and encoding a putative type I polyketide synthase, was dramatically increased (FIG. 24). The enhanced expression of SCO6273 was observed only during vegetative growth. No dre site was predicted upstream of kasO, and the cis-trans relationship between DasR and this cryptic cluster is under investigation.

Involvement of DasR in the Control of Cell Wall Lysis

Chitin is the main form of storage of GlcNAc and the second most abundant polymer on earth, and as such is of immense importance for soil-dwelling bacteria. GlcNAc is a rich N- and C-source and, with its metabolic products acetate, ammonia and fructose-6-P, stands at the crossroads of the major primary metabolic pathways. This underlines the selective advantage of being able to colonise different types of chitin-containing substrates (Saito et al., 2003; Schrempf, 2001). Our experiments suggest that GlcNAc can provide opposite signals, namely expansion (growth and developmental block) under nutrient-rich conditions and growth cessation followed by development (antibiotic production; sporulation) under nutrient-limited conditions. There are two major sources of GlcNAc: chitin and the bacterium's own cell wall, and they may trigger opposite responses. Bacterial chitinases mainly generate chito-oligosaccharides and N,N′-diacetylchitobiose (GlcNAc)₂ from chitin, and little GlcNAc. Also, dasR mutants have a five-fold lower chitinolytic activity than the parental strain (Colson et al., 2007), but overproduce antibiotics, suggesting that chitinases do not produce the signal. The ‘GlcNAc effect’ was observed only at higher concentrations (>5 mM). Perhaps the most likely natural source would be autolysis of the bacterial cell wall. Large amounts of GlcNAc were found to accumulate locally after programmed cell lysis, when general nutrient limitation necessitates development of an aerial mycelium at the expense of the vegetative hyphae (Miguelez et al., 2000). Since we show that nutrient sensing, cell wall lysis and proteolysis and secondary metabolism (in particular antibiotic production) are all linked directly to the function of DasR, there is a highly suggestive clustering within a single regulon of genes involved in the catabolism of peptidoglycan precursors, together with antibiotic pathway-specific activators.

The contrast between the large number of secondary metabolites produced by streptomycetes and the relatively limited knowledge on the global regulatory mechanisms that trigger their production implies that much is to be gained in terms of drug discovery by learning from the organism itself. We propose a signalling cascade from nutrient stress to antibiotic production. Our deduced pathway proposes GlcNAc as an important signalling molecule for streptomycetes, allowing them to determine the nutritional status of the habitat. The signal that is transported by the PTS^(GlcNAc) is metabolized to glucosamine-6-P, inactivating DasR, which in turn is responsible for suppression of antibiotic production and development under nutrient-rich conditions. Besides the PTS^(GlcNAc), DasR controls many more ABC sugar transporters and the functions of several of these are currently under investigation. The observation that antibiotic production can be awakened and/or enhanced by interfering with the DasR-mediated control system opens new perspectives for screening programmes directed at the discovery of novel natural products. Conceivably, the producing potential of thousands of strains could be boosted by addition of GlcNAc, and we have strong evidence that in many cases this makes the difference between a hit and a miss. This will improve the success rate of screening procedures aimed at the discovery of drugs for the treatment of infectious diseases caused by the recurring multi-drug resistant strains (such as MDR- and XDR-Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecalis (VRE)) but also of specific cancers.

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TABLE 1 Experimentally validated DasR binding sites used to build the matrix for consensus sequences. malX2Sco ACTGGTGTAGACCAGT (SEQ ID NO: 11) score = 16.20 nagE2Sco(1) CAAGGTGTAGACCTCT (SEQ ID NO: 12) score = 11.35 nagE2Sco(2) AGTGGTGTAGACCTGT (SEQ ID NO: 13) score = 16.98 nagE2Sco(3) AGTGGTGTAGACCACC (SEQ ID NO: 14) score = 15.01 ptsHSco(1) AGTTGTCTAGACCAGT (SEQ ID NO: 15) score = 15.29 ptsHSco(2) TCTTGTCTAGACCAGT (SEQ ID NO: 16) score = 13.71 crr-ptslSco(1) TGTGGTCTAGACCTCT (SEQ ID NO: 17) score = 15.61 msiKSco GGTGGTGTAGTCCACA (SEQ ID NO: 18) score = 12.52 nagBSco TGTGGTTTAGACCAAT (SEQ ID NO: 19) score = 13.72 nagKASco(1) GGTGGTGTAGACCTTA (SEQ ID NO: 20) score = 13.05 nagKASco(2) AGTGGACTAGACCTCT (SEQ ID NO: 21) score = 14.45 chiFSco(1) AAGGGTGTAGACCAGT (SEQ ID NO: 22) score = 13.55 chiFSco(2) ACTGGTACAGACCAAA (SEQ ID NO: 23) score = 9.73 actII-4 TGTTGAGTAGGCCTGT (SEQ ID NO: 24) score = 11.22

TABLE 2 Known or predicted co-transcribed # Evidence¹ Target gene function dre Pos² Score³ gene⁴ Known or predicted function 1 ERA− SCO2907, PTS EIIC component, ACAGGTCTACACCACT −49 16.98 15.01 — — nagE2 N-acetylglucosamine AGTGGTGTAGACCACC −32 11.35 uptake CAAGGTGTAGACCTCT −236 (SEQ ID NO: 25) 2 SCO2906, PTS EIIC component, not ACTGGTCTACACCAGT −41 16.2 — — nagE1 functional (SEQ ID NO: 26) 3 ERA− SCO2905c, PTS EIIB component, −134 16.2 — — malX2 N-acetylglucosamine uptake 4 E SCO5232, ABC sugar transporter, ACTGGTCTACACCATT −106 15.79 8.15 SCO5233, ABC sugar transporter membrane dasA sugar binding protein CTTGGTCTAGTCCATA −322 dasB/SCO5234, protein/ABC sugar transporter (SEQ ID NO: 27) dasC/SCO5235 membrane protein/putative intracellular Beta-N-acetylglucosaminidase 5 ERA− SCO1390, crr PTS EIIA^(crr) component, TGTGGTCTAGACCTCT −130 15.61 SCO1391, ptsI PTS EI component, N-acetylglucosamine (SEQ ID NO: 28) phosphoenolpyruvate-protein uptake phosphatase 6 E SCO5842 putative acetyl-coenzyme AGTTGTCTAGACCAGT −168 15.29 13.71 — — A synthetase TCTTGTCTAGACCAGT −153 7 ERA− SCO5841c PTS Hpr, (SEQ ID NO: 29) −51, −66 — — N-acetylglucosamine and fructose uptake 8 E SCO4286 ABC sugar transporter, AGAGGTCTAGTCCACT −81. −63 14.45 hypothetical hypothetical protein, unknown sugar binding protein GGTGGTGTAGACCTTA 13.05 function 9 ER− SCO4285c, NagK, (SEQ ID NO: 30)  −83, −101 SCO4284c, nagA NagA, nagK N-acetylglucosamine N-acetylglucosamine-6-phosphate kinase deacetylase 10 E SCO5239 Two-component sensor AGTGGTCTAGTCCACA −335 14.19 — — histidine kinase (SEQ ID NO: 31) 11 ER− SCO5236c, NagB, probable TGTGGTTTAGACCAAT −68 13.72 — — nagB glucosamine phosphate (SEQ ID NO: 32) isomerase 12 E SCO3563, acetoacetyl-coenzyme A ACAGGTCTAAACCATT −102 13.59 — — acsA synthetase (SEQ ID NO: 33) 13 ER+ SCO7263, chiF ChiF chitinase ACTGGTCTACACCCTT −172 13.55 9.73 SCO7264 probable NADPH dependent ACTGGTACAGACCAAA −155 oxidoreductase, (SEQ ID NO: 34) Aldo/keto reductase 14 E SCO7225 secreted chitinase TATGGTCTAGACCTGA −55 13.1812.4611.46 — — TCAGGTCTAGACCTGT −34 CCTTGTCTAGACCAAT −168 15 E SCO7224c possible integral (SEQ ID NO: 35) −272, −293 — — membrane protein, DoxX −159 family, unknown function 16 E SCO1444, chiI ChiI chitinase ACTGGTCTAGTCCTCT −53 12.81 5.22 — — ATTGGTCCATACCTAT −75 (SEQ ID NO: 36) 17 EP− SCO4240c, MsiK, multiple sugar GGTGGTGTAGTCCACA −75 12.52 — — msiK import protein, ABC (SEQ ID NO: 37) transporter ATP-binding protein 18 E SCO5004 hypothetical protein, GGTGGTCCAGACCAAT −258 12.08 — — unknown function (SEQ ID NO: 38) 19 E SCO5003c, ChiA chitinase −77 — — chiA 20 E SCO7056c GntR-family transcriptional ATTGGTCTAAACCAGC −79 12.08 6.85 — — regulator (new subfamily) GCAGGTCTGGTCCTCC −282 (SEQ ID NO: 39) 21 EA− SCO6486, DppA, AGTGGTCCAGACCACC −71 12.03 SCO6487, SCO6488, possible aminoacylase/putative dppA D-alanyl-aminopeptidase (SEQ ID NO: 40) SCO6489, SCO6490 acyl-peptide hydrolase/LD-carboxypeptidase/ putative alanine acetyltransferase 22 E SCO2672 membrane protein, ABC AGAGGTCTGGACAACA −32 11.99 — — transporter, FtsX cell (SEQ ID NO: 41) division permease family, unknown function 23 E SCO2503, chiJ ChiJ putative chitinase AAAGGTCTGGACCACA −78 11.818.47 5.73 — — CTTGGTCCAGACCTCT −99 TCTGGACCACAGCACT −73 (SEQ ID NO: 42) 24 E SCO1429, chiD ChiD chitinase ACTGGTCTAGTCCTCC −96 11.5 5.86 — — AATGGTCCGAACCATT −118 25 E SCO1428c, acd acyl-CoA dehydrogenase (SEQ ID NO: 43) −312 — — −290 26 SCO3679 hypothetical protein, sigma TGTTGTCTAGTCCAAT −314 11.41 — — factor PP2C-like (SEQ ID NO: 44) phosphatase 27 ER− SCO5085, actinorhodin cluster TGTTGAGTAGGCCTGT −59 11.22 — — actII-4 activator protein (SEQ ID NO: 45) 28 E SCO6013 probable AATGGTCTGGACCAGA −274 11.03 8.63 7.95 — — 1-deoxyxylulose-5-phosphate GGTGGACTGGACCACC −201 synthase ATGGGACTAGACCAAT −258 29 E SCO6012c, ChiH chitinase (SEQ ID NO: 46) −111, −184, — — chiH −127 30 SCO4315 possible copper ATTGGACTAGACCTGT −39 10.99 — — homeostasis protein, CutC (SEQ ID NO: 47) family 31 E SCO4671c LysR-family regulatory GCTGGTACAGACCAGT −55 10.83 — — protein (SEQ ID NO: 48) 32 E SCO6300c probable secreted AGAGGTCTAGACAAAA −116 10.67 9.78 — — Beta-N-acetylglucosaminidase ATAGGTCTAGACAAAA −131 (SEQ ID NO: 49) 33 E SCO6005, ABC sugar transporter, AGTGGACTATACCTGT −334 10.56 SCO6006, ABC sugar transporter membrane ngcE sugar binding protein, (SEQ ID NO: 50) ngcF/SCO6007, ngcG protein, NgcF/ABC sugar NgcE transporter membrane protein, NgcG 34 E SCO6004c putative AGTGGACTATACCTGT −244 10.56 — — alpha-1,2-mannosidase (SEQ ID NO: 51) 35 E SCO5376c, ChiC chitinase AAAGGTCTGGACCATA −88 10.35 9.29 SCO5375c possible secreted protein, chic ATAGGTCTGGACCAAT −109 unknown function (SEQ ID NO: 52) 36 E SCO6345 chitinase TAAGGTCTAGACCTGC −114, −94 9.99 8.74 — — 37 E SCO6344c putative secreted amidase GTAGGTCTAGACCTGC −133, −153 — — (SEQ ID NO: 53) 38 SCO1212 putative Mur_like ligase TGAGGTCCACACCACG −76 9.92 SCO1213 conserved hypothetical protein 39 SCO1211c putative polypeptide (SEQ ID NO: 54) −5 — — deformylase 40 SCO1083c putative flavin-dependent TGTGGAGAAGACCTCA −129 9.48 — — reductase (SEQ ID NO: 55) 41 E SCO1433 hypothetical protein, ATTGGTGTCGACCACT −205 9.41 — — unknown function (SEQ ID NO: 56) 42 E SCO1432c possible membrane ATTGGTGTCGACCACT −86 SCO1431c possible membrane protein, protein, unknown function (SEQ ID NO: 57) unknown function 43 SCO5266 putative membrane protein CATGGTGCAGACCTCC −139 9.25 — — (SEQ ID NO: 58) 44 SCO5265c hypothetical protein CATGGTGCAGACCTCC 38 9.25 SCO5264c hypothetical protein SC7G11.26c (SEQ ID NO: 59) 45 E SCO0481, putative secreted chitin TATGGTCTAGTCCAAC −201 9.19 — — chb3 binding protein (SEQ ID NO: 60) 46 E SCO2753 Lacl-family transcriptional GGTGGTCTGGACAAGA −120 9.15 — — regulator, NagR (SEQ ID NO: 61) 47 E SCO2752c possible oxidoreductase, −127 SCO2751c, hypothetical protein/putative unknown SCO2750c isomerase, unknown function 48 E SCO7250c putative AGTGGCGTACACCTGT −213 9.04 — — N-acetylmuramoyl-L-alanine (SEQ ID NO: 62) amidase 49 E SCO5673, chiB ChiB chitinase ATTGGTCTGGACCAAA −63 9.03 — — (SEQ ID NO: 63) 50 SCO7699 putative nucleotide-binding GAGGGTCCAGACCTCT −245 9.0 SCO7700/SCO7701 putative cyclase/putative protein, (SEQ ID NO: 64) methyltransferase sporulation-specific protein p3 (S. griseus) 51 SCO7698c putative merR-family −19 — — transcriptional regulator 52 E SCO2833c, chb secreted chitin binding GCAGGTCTAGACCAAG −70 8.91 — — protein (SEQ ID NO: 65) 53 E SCO2946c ABC sugar transporter, AGAGGTCTGAACCAAT −112 8.91 SCO2945c, ABC sugar transporter membrane sugar binding protein (SEQ ID NO: 66) SCO2944c, protein, ABC sugar transporter SCO2943c membrane protein, putative intracellular beta-N-acetylglucosaminidase 54 E SCO1117c putative CGCGGTCTAGACCAAA −131 8.79 — — 3-carboxymuconate (SEQ ID NO: 67) cyclase 55 E SCO5230c integral membrane protein, TCTGGTCTAGTCCTGG −118 8.77 SCO5229c probable permease, putative sensory protein (SEQ ID NO: 68) sodium:solute symporter family 56 SCO6149 putative ATP GTP-binding GGAGGTGTCGACCAAT −140 8.76 SCO6150, CO6151 putative ADA-like regulatory protein (SEQ ID NO: 69) protein/putative methylated-DNA-protein-cysteine methyltransferase 57 SCO6319 putative lipoprotein ATTGGTCTGAACCATG −30 8.76 — — (SEQ ID NO: 70) 58 SCO6033 hypothetical protein CTTGGTCTAGTCCATT −278 8.68 — — SC1C3.21 (SEQ ID NO: 71) 59 SCO6032c beta-N-acetylglucosaminidase −154 — — 60 E SCOEc, chiE ChiE chitinase and CTTGGTCCAGACCTGT −188 8.65 — — metallopeptidase (SEQ ID NO: 72) 61 SCO4394, iron repressor TGCGGTCTGGACCAGT −184, +9 8.42 7.45 — — desR ACTGATCGACACCACG 62 SCO4393c Possible phosphosugar (SEQ ID NO: 73) −247, −55, — — isomerase 63 SCO6084 putative DNA polymerase GAGGGTGGAGACCACT −292, −49 8.3 8.13 — — GGTGGTGCAGTCCTAC (SEQ ID NO: 74) 64 SCO5046, wb/l hypothetical protein TCAGGAGTAGACCCGT −14 8.23 — — (SEQ ID NO: 75) 65 SCO1954 hypothetical protein GGATGTGAAGACCTCT −101 8.15 — — 66 SCO1953c ABC excision nuclease (SEQ ID NO: 76) −268 SCO1952c, hypothetical protein/conserved subunit C SCO1951c, hypothetical protein/ SCO1950c hypothetical protein 67 SCO5231c, DasR, gntR-family CTTGGTCTAGTCCATA −150 8.15 — — dasR transcriptional regulator (SEQ ID NO: 77) 68 SCO4506 conserved hypothetical AGAGGTCAAGATCACT −103 8.05 SCO4507 putative serine/threonine protein (SEQ ID NO: 78) protein kinase 69 SCO4505c, cold shock protein −206 — — scoF2 70 SCO3152c hypothetical protein AGTGGACTCCTCCACC −50 8.03 — — SCE87.03c (SEQ ID NO: 79) 71 SCO6232 putative beta-mannosidase TCAGGACTAGACCGGT −86 7.97 SCO6233 putative transcriptional regulator 72 SCO6231c probable sugar transport (SEQ ID NO: 80) −202 — — system sugar-binding lipoprotein SC2H4.13c 73 E SCO1906c putative secreted protein, ACTGGCGGAGACCTCT −128 7.93 — — unknown function (SEQ ID NO: 81) 74 SCO2119c, 6-phosphofructokinase GGTGGTTGAGGCCACT −40 7.83 — — pfkA (SEQ ID NO: 82) 75 SCO1971 conserved hypothetical TGTGGTCGAGACGTGT −172 7.77 SCO1972 putative sugar kinase protein (SEQ ID NO: 83) 76 SCO1970c putative dioxygenase 30 — — 77 SCO1289 putative gntR-family CGTGGTGCAGACGTGA −36 7.73 — — regulatory protein (SEQ ID NO: 84) 78 SCO1288c putative integral −143 SCO1287c hypothetical protein membrane protein 79 SCO0073 hypothetical protein CCAGGTTCAGACCTGT −219 7.69 SCO0074, SCO0075 hypothetical protein/ (SEQ ID NO: 85) hypothetical protein 80 SCO0072c putative wall associated −309 — — protein 81 SCO5463 putative MerR-family ACTGGCCCGCACCACC 39 7.55 SCO5464 — transcriptional regulator (SEQ ID NO: 86) 82 SCO5462c hypothetical protein −103 — — SC3D11.19c 83 SCO5016c putative integral GGTGGAGCAGACCGGA −280 7.5 — — membrane protein (SEQ ID NO: 87) 84 E SCO3975c putative regulator TGTGGTCGAGACCGGA −86 7.49 — — (SEQ ID NO: 88) 85 SCO5366, atpl ATP synthase protein I AGAGGTAAAGACCTCA −172 7.49 — — (SEQ ID NO: 89) 86 SCO2787 conserved hypothetical ACGGGTGCGGACCACT −61 7.44 SCO2788, SCO2789, hypothetical protein protein SCC105.18 (SEQ ID NO: 90) glmS2 SCC105.19/glucosamine- fructose-6-phosphate aminotransferase 87 SCO2786c beta-N-acetylhexosaminidase −70 — — 88 SCO4442 hypothetical protein ATTGGCGTAAACCACA −41 7.41 — — SCD6.20 (SEQ ID NO: 91) 89 SCO1752 putative integral TGTGGCATGCACCACT −80 7.29 — — membrane protein (SEQ ID NO: 92) 90 SCO1751c putative transmembrane −198 — — transport protein 91 SCO6003c putative DNA-binding GCCGGTGAAGACCAGT −235 7.26 — — protein (SEQ ID NO: 93) 92 E SCO5716c putative peptide transport ATTGGCGCAGACCACT −197 7.24 — — system secreted peptide (SEQ ID NO: 94) binding protein 93 SCO5330 hypothetical protein GCTGGCGTAGCCCACT −54 7.16 — — SC6G9.03c (SEQ ID NO: 95) 93 SCO5430c putative extracellular AATGGTCTAGTCAGGT −81 7.02 SCO5429c, putative integral membrane solute-binding lipoprotein (SEQ ID NO: 96) SCO5428c transport protein/putative integral membrane transport protein 94 SCO2685c putative ATP-binding AGTGGACAACACCCGA −142 6.95 SCO2684c putative ATP-binding protein SCC61A.06c (SEQ ID NO: 97) membrane protein 96 E SCO4516c hypothetical protein ACTGGTCTGGATCCGT −20 6.91 SCO4515c putative membrane protein SCD35.23c (SEQ ID NO: 98) 97 SCO7054 conserved hypothetical TGTGGAGTAGAGTAGT −47 6.89 SCO7055 putative methyltransferase protein (SEQ ID NO: 99) 98 SCO7053c hypothetical protein −50 — — 99 E SCO4735 30S ribosomal protein S9 CGTGGCCGAGACCACT −1 6.88 — — (SEQ ID NO: 100) 100 SCO4722, preprotein translocase GCTCGTCTGAACCACT −266 6.87 SCO4723 adenylate kinase secY SecY subunit (SEQ ID NO: 101) 101 SCO7509c conserved hypothetical GCGGGTGAAGACCAGC 20 6.83 — — protein (SEQ ID NO: 102) 102 SCO4646, preprotein translocase ACTGGTCTCCAAAACC −156 6.81 SCO4647 transcription antitermination secE SecE subunit (SEQ ID NO: 103) protein 103 SCO4645c aspartate ACTGGTCTCCAAAACC −281 6.81 — — aminotransferase (SEQ ID NO: 104) 104 SCO4562 NuoA, NADH GGTGGTGGAGATCACA −206 6.78 SCO4563-SCO4575 NADH dehydrogenase subunits dehydrogenase subunit (SEQ ID NO: 105) (nuoBCDEFGHIJKLM NuoA-NuoN N) 105 SCO1692c putative oxidoreductase CGCGGTCTACTCCATT −116 6.76 SCO1691c putative tetR transcriptional (SEQ ID NO: 106) regulator 105 SCO4904c putative integral TGAGGCCTGCGCCACA −72 6.69 — — membrane protein (SEQ ID NO: 107) 107 SCO5954 chitinase (putative ATTGGTCCAGACCTTC −95 6.64 — — secreted protein) (SEQ ID NO: 108) 108 SCO5805, nrdJ ribonucleotide reductase AGTGAACAAGACCTGT −117 6.61 — — (SEQ ID NO: 109) 109 SCO4609 putative peptidase CCTGGCGTTGACCAGT −136 6.6 SCO4610/SCO4611 putative integral membrane (SEQ ID NO: 110) protein/hypothetical protein SCD39.11 110 SCO2547c putative hydrolase GGTGGTCCGGTCCTGT −24 6.58 SCO2546c probable adenosine deaminase (SEQ ID NO: 111) 111 SCO4963 putative ABC transporter CCTGGTGAAGACCTTC 27 6.55 SCO4964 putative integral membrane ATP-binding protein (SEQ ID NO: 112) transport protein 112 SCO2037c, tryptophan synthase beta GGTGGATCAGACCGCT −69 6.54 SCO2036c tryptophan synthase alpha trpB subunit (SEQ ID NO: 113) subunit 113 SCO0915 hypothetical protein TTTGGTATGGACCATT −98 6.52 — — SCM10.03 (SEQ ID NO: 114) 114 SCO0914c putative lipoprotein −127 — — 115 SCO2802 putative secreted protein AGAGGACTCGTCCACG 16 6.51 — — (SEQ ID NO: 115) 116 SCO6914 hypothetical protein GGTGCTGGAGACCTCA −133 6.51 SCO6915 hypothetical protein SC1B2.21 SC1B2.20 (SEQ ID NO: 116) 117 E SCO5609c hypothetical protein ACTGGTCAGGACCGCT −133 6.38 — — SC2E1.26c (SEQ ID NO: 117) 118 SCO3261 putative ATP-binding ACCGGGCTACACACCT −284 6.37 SCO3262 hypothetical protein protein (SEQ ID NO: 118) 119 SCO5636c, transcriptional regulator ACTGGACGAGACCCCG −187 6.34 — — korSA (SEQ ID NO: 119) 120 E SCO7070 chitosanase ACAGGTCCGGACCAAT −50 6.34 — — (SEQ ID NO: 120) 121 E SCO7069c chitinase −61 — — 122 SCO1558c putative ABC transporter GGAGGCCTGCTCCAGC 11 6.32 — — permease protein (SEQ ID NO: 121) 123 SCO5717c conserved hypothetical CCGGGTGTAGCCCAGC 31 6.32 — — protein SC3C3.03c (SEQ ID NO: 122) 124 SCO3510c putative DNA methylase CGGGGTCTGGACCTGC −42 6.29 — — (SEQ ID NO: 123) 125 SCO4811 putative integral CGTGATCCAGACCACC 9 6.25 — — membrane protein (SEQ ID NO: 124) 126 SCO3560 putative ATP-binding AGTGGTCTTCTTCACC −61 6.24 — — protein (SEQ ID NO: 125) 127 SCO3559c putative oxidoreductase −58 — — 128 SCO5276 conserved hypothetical ACGGGTCCACTCAACA −56 6.24 SCO5277, SCO5278, putative magnesium protein (SEQ ID NO: 126) SCO5279, SCO5280 chelatase/putative magnesium chelatase/hypothetical protein SCCB12.03/putative ATP-binding protein 129 R− SCO5881c, Undecylprodigiosine AGTGGTTTCCACCTCA −201 6.24 — — redZ activator (SEQ ID NO: 127) 130 SCO1262c putative gntR-family CTTGGTCAAGACCAAT −113 6.22 — — transcriptional regulator (SEQ ID NO: 128) 131 SCO2141 putative small secreted ATCGGCCTGGACAACT −167 6.19 SCO2142, SCO2143, putative two component sensor hydrophilic protein (SEQ ID NO: 129) SCO2144, SCO2145 kinase/putative two component system response regulator/ putative integral membrane transporter/putative glycerate kinase ¹If confirmed target, method is indicated: E, EMSA (target site bound by DasR), A, enzyme Assay, R, RT PCR. “+” means shown to be activated by DasR, “−”, shown to be repressed by DasR. ²Position is last nucleotide position of the dre target site relative to the translational start site of the gene. ³Weighed matrix score of the dre site. Cut-off of 6.00 was taken as bottom limit. Higher score means better fit to the consensus sequence. ⁴If genes are known or predicted to be in an operon (generally because there is no or almost no intergenic region), the presumed co-transcribed genes are shown.

TABLE 3 List of putative binding sites for DasR relating to secondary metabolism (cut-off score 5) A. Antibiotics and metabolites of known function produced by actinomycetes Secondary metabolite Streptomyces strain dre sequence Target Function score Clavulanic acid S. clavuligerus attggagtagacctct (SEQ ID NO: 130) pcbR PBP; β-lactam resistance 11.47 ggagggctggaccagc (SEQ ID NO: 131) pcbR PBP; β-lactam resistance 5.72 Actinorhodin S. coelicolor tgttgagtaggcctgt (SEQ ID NO: 132) actII-ORF4 pathway-specific activator 11.22 Undecylprodigiosin S. coelicolor gcaggtggagaccacc (SEQ ID NO: 133) redZ pathway-specific activator 6.61 tgaggtggaaaccact (SEQ ID NO: 134) redZ pathway-specific activator 6.24 Valanimycin S. viridifaciens ctctgagtaggcctgt (SEQ ID NO: 135) vlmM Valanimycin transferase 8.01 Daptomycin S. filamentosus actggtgtcgaccagc (SEQ ID NO: 136) dptD peptide synthetase 3 10.12 ggaggtcgagaccagt (SEQ ID NO: 137) dptP hypothetical 9.24 tgaggtgtacgccacc (SEQ ID NO: 138) AAX31564 Putative phosphatase 6.59 tgaggtcgaggccacc (SEQ ID NO: 139) dptD peptide synthetase 3 5.98 agtggtgctgcccaat (SEQ ID NO: 140) dptP hypothetical 5.97 cgtgatctacacctcc (SEQ ID NO: 141) dptBC peptide synthetase 2 5.91 cgtgatctacacctcc (SEQ ID NO: 142) dptBC peptide synthetase 2 5.91 acaagtccacaccccc (SEQ ID NO: 143) dptA peptide synthetase 1 5.53 cgaggcgtagacctgg (SEQ ID NO: 144) AAX31565 metalloprotease 5.43 cgtggcctggacctca (SEQ ID NO: 145) dptBC peptide synthetase 2 5.39 ggtggtcctcaccacg (SEQ ID NO: 146) dptBC peptide synthetase 2 5.33 actggagtccacctga (SEQ ID NO: 147) AAX31520 ATP-dependent helicase 5.00 cgaggcctacaccctc (SEQ ID NO: 148) AAX31577 hypothetical 6.05 Novobiocin S. spheroides ggaggtgtagatcaca (SEQ ID NO: 149) novH Peptide synthase 8.45 Actinomycin S. anulatus ggaggtgtagatcaca (SEQ ID NO: 150) acmC peptide synthase III 8.45 acmC peptide synthase III A47934 S. toyocaensis ggaggtgtagatcaca (SEQ ID NO: 151) staC peptide synthetase 8.45 (teichoplanin) tctggtggagaccttc (SEQ ID NO: 152) staP membrane protein 7.73 actggtctgctcgatg (SEQ ID NO: 153) staN ion transporter 6.23 gctggtccaggcccct (SEQ ID NO: 154) dpgB enhancer of DihydroxyphenylAcCoA synthetase activity 6.21 ggtggtctccagcacc (SEQ ID NO: 155) vanSst histidine kinase 5.87 gctggtctcgaccctc (SEQ ID NO: 156) vanSst histidine kinase 5.76 catggtctcgtccagc (SEQ ID NO: 157) staF P450-related oxidase 5.06 Streptomycin S. griseus gggggtgtcgaccagc (SEQ ID NO: 158) CAH94374 hypothetical 7.81 cgtggcgcagtccaca (SEQ ID NO: 159) CAH94409 methyltransferase 5.74 cctggtcttcaccccg (SEQ ID NO: 160) strZ transmembrane protein 5.66 agtggtctgcgcatgc (SEQ ID NO: 161) apbE thiamine biosynthesis lipoprotein 5.13 tgacgtctactccttc (SEQ ID NO: 162) CAH94395 PqrB-type multidrug efflux protein 5.03 ggtggggcagaccatc (SEQ ID NO: 163) strB1 Amidinotransferase I 6.22 agttgtgcagacgggt (SEQ ID NO: 164) strZ transmembrane protein 6.09 attggcctgcaccgcg (SEQ ID NO: 165) strU NAD(P) dependent oxidoreductase 6.04 Chloramphenicol S. venezuelae acgggtctacacctcc (SEQ ID NO: 166) papD p-aminobenzoic acid synthase ORFIV 10.38 gctggtgtcgaccatc (SEQ ID NO: 167) papA 4-amino-4-deoxychorismate 7.34 synthase cgaggtggagacctac (SEQ ID NO: 168) papA 4-amino-4-deoxychorismate 5.91 synthase cgaggtggagacctac (SEQ ID NO: 169) papAB p-aminobenzoate synthase Butyrolactone S. virginiae actggtgtcgaccag (SEQ ID NO: 170) barB hormone-like γ-butyrolactone 10.12 biosynthesis B. Known and cryptic biosynthesis clusters of Streptomyces coelicolor metabolite gene function dre position score co-transcribed function actinorhodin SCO5085 actinorhodin cluster TGTTGAGTAGGCCTGT −59 11.22 — — activator protein (SEQ ID NO: 171) actinorhodin SCO5090 actinorhodin GGTGGTCCACACCCTG 24 5.58 SCO5091/SCO5092 cyclase/actinorhodin polyketide putative polyketide synthase (SEQ ID NO: 172) dimerase bifunctional cyclase/ dehydratase Prodigiosin SCO5879 acyl-coa dehydrogenase ACAGGTCTACGGCACG −324 7.33 SCO5880 RedY protein RedW (SEQ ID NO: 173) Prodigiosin SCO5881c RedZ response AGTGGTTTCCACCTCA −201 6.24 — — regulator (SEQ ID NO: 174) Prodigiosin SCO5883c hypothetical protein GTTGGCCTGCTCCAGG −252 5.11 — — SC3F7.03c (SEQ ID NO: 175) CDA SCO3237c conserved hypothetical GTAGGTCTCGACCTCC −151 5.84 — — (calcium-dependent protein (SEQ ID NO: 176) antibiotic) CDA SCO3226 two component system AGCGGTCTGCTCGACT −99 5.75 — — (calcium-dependent response regulator (SEQ ID NO: 177) antibiotic) CDA SCO3234 putative CGATGTCCAGACCGGT 15 5.51 — — (calcium-dependent phosphotransferase (SEQ ID NO: 178) antibiotic) Isorenieratine SCO0188 putative GCAGGACTACACCGTG −168 5.6 — — methylesterase (SEQ ID NO: 179) Tetrahydroxynaphtalene SCO1206 putative polyketide GCTGGTGGAGACCGGC −274 5.6 SCO1207/SCO1208 putative cytochrome P450/hypothetical protein (melanine) synthase (SEQ ID NO: 180) Unknown SCO0387 putative bi-domain GGTTGTGCAGAACTAC −4 5.04 SCO0388/SCO0389/ hypothetical protein SCF62.14/putative oxidoreductase (SEQ ID NO: 181) SCO0390/SCO0391/ lipoprotein/putative membrane protein/putative SCO0392/SCO0393/ transferase/putative methyltransferase/ SCO0394/SCO0395/ putative transferase/hypothetical protein SCO0396/SCO0397/ SCF62.20/putative epimerase/dehydratase/ SCO0398/SCO0399/ hypothetical protein SCF62.22/putative SCO0400/SCO0401 integral membrane protein/putative glycosyl transferase/putative membrane protein/ putative epimerase/putative aminotransferase Unknown SCO0388 hypothetical protein GCTGGTCGCCACCACG −87 5.51 SCO0389/SCO0390/ putative lipoprotein/putative membrane SCF62.14 (SEQ ID NO: 182) SCO0391/SCO0392/ protein/putative transferase/putative SCO0393/SCO0394/ methyltransferase/putative transferase/ SCO0395/SCO0396/ hypothetical protein SCF62.20/putative SCO0397/SCO0398/ epimerase/dehydratase/hypothetical protein SCO0399/SCO0400/ SCF62.22/putative integral membrane SCO0401 protein/putative glycosyl transferase/putative membrane protein/putative epimerase/ putative aminotransferase Unknown SCO6282c putative GCTGGACGAGTCCACC −262 5.42 — — 3-oxoacyl-[acyl- (SEQ ID NO: 183) carrier protein] reductase

TABLE 4 DasR target genes related to glutamate metabolism. Targets, validation (experimental or in silico) and gene function are presented. For meaning and deduction of the score, see text. # Gene Known or predicted gene product Evidence dre Score 1 tRNA Gln tRNA Gln anticodon CTG. S ACTGGTCTAAACCACA (SEQ ID NO: 184) 14.43 2 tRNA Glu tRNA Glu anticodon CTC. S ACTGGTCTAAACCACA (SEQ ID NO: 185) 14.43 3 SCO4285c, N-acetylglucosamine kinase. S, E AGAGGTCTAGTCCACT GGTGGTGTAGACCTTA (SEQ ID NO: 186) 12.82 8.00 nagK ATP + N-acetyl-D-glucosamine = ADP + N-acetyl-D-glucosamine- 6-phosphate 4 SCO4284c, N-acetylglucosamine-6-phosphate deacetylase. S, E, P AGAGGTCTAGTCCACT GGTGGTGTAGACCTTA (SEQ ID NO: 187) 12.82 8.00 nagA CoA + N-acetyl-D-glucosamine 6-phosphate = acetyl-CoA + D-glucosamine-6-phosphate 5 SCO5236c, Glucosamine-6 phosphate isomerase. S, E TGTGGTTTAGACCAAT (SEQ ID NO: 188) 11.36 nagB L-glutamine + D-fructose 6-phosphate = L-glutamate + D-glucosamine-6-phosphate 6 SCO6344 Glu-tRNAGln amidotransferase A subunit. S TAAGGTCTAGACCTGC (SEQ ID NO: 189)  9.99 ATP + glutamyl-tRNA(Gln) + L-glutamine = ADP + phosphate + glutaminyl-tRNA(Gln) + L-glutamate 7 SCO5520 pyrroline-5-carboxylate dehydrogenase. P — — 1-pyrroline-5-carboxylate + NAD+ + H2O = L-glutamate + NADH + H+ 8 SCO4683 GdhA, NADP-specific glutamate dehydrogenase. P — — L-glutamate + H2O + NAD+ = 2-oxoglutarate + NH3 + NADH + H+ 9 SCO4366 phosphoserine aminotransferase. P — — O-phospho-L-serine + 2-oxoglutarate = 3-phosphonooxypyruvate + L-glutamate S, predicted in silico; E, experimentally validated in vitro; P, deduced from proteomics experiments; dre, DasR responsive element. Scores are expressed in unit of bits.

TABLE 5 DasR binding sites in Bacillus species. Score Position Site Gene Synonym COG Product A. B. subtilis 4.70572 −121 AGTGATCTATACCAAT (SEQ ID NO: 190) yflG Bsu0769 COG0024 similar to methionine aminopeptidase 4.70572 −67 ATTGGTATAGATCACT (SEQ ID NO: 191) yflF Bsu0770 COG1264 similar to phosphotransferase system enzyme II 4.54507 −60 AACGGTCTAGACAAAT (SEQ ID NO: 192) yxaG Bsu3995 — yxaG 4.47401 −127 AGTGATCTAGACCAGC (SEQ ID NO: 193) yvoB Bsu3497 COG1493 similar to hypothetical proteins 4.47401 −71 GCTGGTCTAGATCACT (SEQ ID NO: 194) nagA Bsu3498 COG1820 N-acetylglucosamine-6-phosphate deacetylase 4.41253 −44 AGTTGTATATACAAGT (SEQ ID NO: 195) treP Bsu0780 COG1264 phosphotransferase system (PTS) trehalose-specific enzyme IIBC component 4.41253 −165 ACTTGTATATACAACT (SEQ ID NO: 196) yfkQ Bsu0779 — similar to spore germination response 4.3857 −174 ATATGTATAGACCTGT (SEQ ID NO: 197) yqjU Bsu2373 — yqjU 4.3246 −326 ATCTGTCTATACCTAT (SEQ ID NO: 198) yomE Bsu2140 — yomE 4.28564 −82 AATAGTATAGACTATT (SEQ ID NO: 199) pckA Bsu3051 COG1866 phosphoenolpyruvate carboxykinase 4.25776 −320 TTTTGTATATACCATT (SEQ ID NO: 200) ydbO Bsu0454 COG0053 similar to hypothetical proteins 4.25776 −61 AATGGTATATACAAAA (SEQ ID NO: 201) ydbN Bsu0453 — ydbN 4.25487 −68 AGTGGTCTAAACTCCT (SEQ ID NO: 202) bofA Bsu0023 — integral membrane protein 4.24264 −226 AATGGTATATATCATA (SEQ ID NO: 203) yodL Bsu1963 — yodL 4.23975 −346 ATGGCTCTACACCATT (SEQ ID NO: 204) ylaD Bsu1476 — ylaD 4.23813 −2 AATGGAATATACCAGT (SEQ ID NO: 205) yhdN Bsu0953 COG0667 similar to aldo/keto reductase 4.23813 −211 ACTGGTATATTCCATT (SEQ ID NO: 206) yhdM Bsu0952 COG1595 similar to RNA polymerase ECF-type sigma factor 4.23131 −190 ATTGGTTTAGACAACA (SEQ ID NO: 207) tenI Bsu1167 COG0352 transcriptional regulator 4.23065 −91 ACATTTCTATACCATT (SEQ ID NO: 208) hemE Bsu1012 COG0407 uroporphyrinogen III decarboxylase 4.20249 −113 AATTATATATACAATT (SEQ ID NO: 209) rbsR Bsu3589 COG1609 transcriptional regulator (LacI family) 4.20249 −46 AATGTTATATAACATT (SEQ ID NO: 210) yurO Bsu3257 COG1653 similar to multiple sugar-binding protein 4.20249 −115 AATGGTATATAATATT (SEQ ID NO: 211) yndL Bsu1783 — similar to phage-related replication protein 4.19036 −170 ATTCGTATAAACAAGT (SEQ ID NO: 212) yeeA Bsu0677 COG1002 similar to hypothetical proteins 4.19036 −10 AATGGTTTATATGAAT (SEQ ID NO: 213) yqeK Bsu2559 COG1713 similar to hypothetical proteins 4.19036 −237 ACTTGTTTATACGAAT (SEQ ID NO: 214) yefB Bsu0675 — similar to site-specific recombinase B. B. halodurans 4.62842 −168 ATTTGTATATACCAAT (SEQ ID NO: 215) BH0422 BH0422 COG1263 PTS system, N-acetylglucosamine-specific enzyme II, ABC component 4.62842 −88 ATTGATATATACCAAT (SEQ ID NO: 216) BH3323 BH3323 COG2188 transcriptional regulator (GntR family) 4.62842 −155 ATTGGTATATACAAAT (SEQ ID NO: 217) nagA BH0421 COG1820 N-acetylglucosamine-6-phosphate deacetylase 4.62842 −231 ATTGGTATATATCAAT (SEQ ID NO: 218) BH3324 BH3324 COG1208 glucose-1-phosphate thymidylyltransferase 4.54325 −152 ATTGGTATAGACATTT (SEQ ID NO: 219) BH0419 BH0419 COG2188 transcriptional regulator (GntR family) 4.54325 −103 AAATGTCTATACCAAT (SEQ ID NO: 220) BH0418 BH0418 COG0500 BH0418~unknown conserved protein in others 4.5403 −155 TATGGTATAGACCACT (SEQ ID NO: 221) BH2230 BH2230 — BH2230~unknown 4.50391 −214 ATTGGTATAAACAAAT (SEQ ID NO: 222) BH1924 BH1924 COG1653 sugar transport system (sugar-binding protein) 4.48969 −175 ATTCGTTTAGACCAAT (SEQ ID NO: 223) BH0593 BH0593 — BH0593~unknown 4.45248 −41 ATTGTTCTAGACCCTT (SEQ ID NO: 224) BH2561 BH2561 COG3879 BH2561~unknown conserved protein in bacilli 4.41889 −63 ACTTGTATATACAAAT (SEQ ID NO: 225) BH2216 BH2216 COG1264 PTS system, trehalose-specific enzyme II, BC component 4.41579 −316 AATGGTCTACACCAAG (SEQ ID NO: 226) BH1484 BH1484 COG3595 BH1484~unknown conserved protein in others 4.41579 −302 CTTGGTGTAGACCATT (SEQ ID NO: 227) BH1482 BH1482 COG0517 BH1482~unknown conserved protein in B. subtilis 4.40381 −99 ACTTGTATATACAAGT (SEQ ID NO: 228) treA BH0872 COG0366 alpha,alpha-phosphotrehalase 4.40367 −81 AAAGGTGTAGATCATT (SEQ ID NO: 229) BH0661 BH0661 — response regulator aspartate phosphatase 4.37739 −104 AATGATTTAGATCAAT (SEQ ID NO: 230) BH0786 BH0786 COG1940 transcriptional regulator 4.33078 −136 TATGGTCTATATCATT (SEQ ID NO: 231) BH0464 BH0464 COG1968 bacitracin resistance protein (undecaprenol kinase) 4.29237 −81 AATAGTATAGACTATT (SEQ ID NO: 232) pckA BH3302 COG1866 phosphoenolpyruvate carboxykinase 4.25057 −344 ACAGGTGTAGACATTT (SEQ ID NO: 233) BH3024 BH3024 COG0745 BH3024~unknown 4.22134 −148 AGTTGTTTAGACCAGA (SEQ ID NO: 234) BH2314 BH2314 COG0191 fructose bisphosphate aldolase 4.21655 −71 ATTGATCTATACAAAC (SEQ ID NO: 235) BH3244 BH3244 — general stress protein 4.21022 −216 ATTGATATAGATGAGT (SEQ ID NO: 236) BH2450 BH2450 — BH2450~unknown 4.19716 −314 AATGGTGTATAGAAAT (SEQ ID NO: 237) BH3530 BH3530 — BH3530~unknown 4.19716 −145 ATTGATTTATAGCATT (SEQ ID NO: 238) BH4039 BH4039 COG0582 BH4039~unknown conserved protein 4.1957 −3 ACATGTCTATACATCT (SEQ ID NO: 239) BH3678 BH3678 COG2972 two-component sensor histidine kinase 4.16786 −302 AATGGTGTAGAGGATT (SEQ ID NO: 240) rpoB BH0126 COG0085 DNA-directed RNA polymerase beta subunit 4.15636 −186 ATTGGTTTATATATAT (SEQ ID NO: 241) BH2699 BH2699 COG1136 ABC transporter (ATP-binding protein) 4.13718 −216 ATTGATCTAGAGCATA (SEQ ID NO: 242) spoVFA BH2403 — dipicolinate synthase subunit A 4.13468 −241 ATCGGTTTACACAATT (SEQ ID NO: 243) rplC BH0134 COG0087 50S ribosomal protein L3

TABLE 6 DasR binding sites in Lactococcus lactis Score Position Site Gene Synonym COG Product 5.86694 −36 ATTGATATATACCAAT (SEQ ID NO: 244) nagB L14408 COG0363 glucosamine-6-P isomerase (EC 5.3.1.10) 5.58801 −72 ATTGGTATATACTGTT (SEQ ID NO: 245) nagA L173068 COG1820 N-acetylglucosamine-6- phosphate deacetylase (EC 3.5.1.25) 5.4239 −40 ATTGGTATATAAAAAT (SEQ ID NO: 246) yxfB L141634 COG0500 HYPOTHETICAL PROTEIN 5.3542 −215 AACGGTATATACGATT (SEQ ID NO: 247) yveE L127921 — UNKNOWN PROTEIN 5.24344 −173 AACAGTATATATCATT (SEQ ID NO: 248) pi228 L51784 — prophage pi2 protein 28 5.18192 −37 AGTGGTATATATTGTT (SEQ ID NO: 249) rgrB L0151 COG2188 GntR family transcriptional regulator 5.16551 −279 AATGATATATATCTTT (SEQ ID NO: 250) ymgC L61341 COG2936 conserved hypothetical protein 5.11627 −170 ACTTGTATATACTTAT (SEQ ID NO: 251) rplJ L0407 COG0244 50S ribosomal protein L10 5.03205 −164 ATGGGTAGATAACAAT (SEQ ID NO: 252) pi234 L57508 — prophage pi2 protein 34 5.03205 −1 AATGAGATATATCAAT (SEQ ID NO: 253) zitQ L166512 COG1121 zinc ABC transporter ATP binding protein 4.95193 −261 ATTGGTTTATACCGAC (SEQ ID NO: 254) dexC L128694 COG0366 neopullulanase (EC 3.2.1.135) 4.95193 −123 GTCGGTATAAACCAAT (SEQ ID NO: 255) malE L128695 COG2182 maltose ABC transporter substrate binding protein 4.92129 −265 ATTGGTATACAATATT (SEQ ID NO: 256) yviA L163025 COG2323 HYPOTHETICAL PROTEIN 4.92129 −118 ATTAGTCTATATCTAT (SEQ ID NO: 257) tra983B L0444 COG2826 transposase of IS983B 4.92129 −189 ATAAATAAATACCAAT (SEQ ID NO: 258) yrjB L174321 COG0247 oxidoreductase 4.91311 −348 AATGGGATATACTGGT (SEQ ID NO: 259) yqeL L22900 COG1161 GTP-binding protein 4.89476 −286 ATTGATATATATGTCT (SEQ ID NO: 260) ycbC L11986 — HYPOTHETICAL PROTEIN 4.89476 −314 ACGAGTATATATAAAT (SEQ ID NO: 261) yliA L179789 — positive transcriptional regulator 4.88629 −220 ATTGGTATAGGTCAAT (SEQ ID NO: 262) ybhE L176316 COG3589 HYPOTHETICAL PROTEIN 4.88246 −50 ATAAGTATATACATCT (SEQ ID NO: 263) yfhF L174076 — HYPOTHETICAL PROTEIN 4.8617 −262 AATGGAAGATACCATT (SEQ ID NO: 264) ywaH L191704 — UNKNOWN PROTEIN 4.85976 −241 ACTTGTATTTATCAAT (SEQ ID NO: 265) ps208 L106731 — prophage ps2 protein 08

TABLE 7 DasR binding sites in Streptococcus species Score Position Site Gene Synonym COG Product A. S. pneumoniae TIGR4 4.56012 −264 AGTGGTGTATGCCAAT (SEQ ID NO: 266) — SP0571 COG2184 cell filamentation protein Fic-related protein 4.52678 −49 ATTGGTCTATACCATA (SEQ ID NO: 267) — SP1415 COG0363 glucosamine-6-phosphate isomerase 4.52678 −118 TATGGTATAGACCAAT (SEQ ID NO: 268) — SP1416 COG0809 S-adenosylmethionine:tRNA ribosyltransferase-isomerase 4.50546 −56 ATTAGACTATACCAAT (SEQ ID NO: 269) — SP0266 COG0449 glucosamine--fructose-6-phosphate aminotransferase, isomerizing 4.45088 −9 AGTGGAATATGACAGT (SEQ ID NO: 270) — SP0856 COG0115 branched-chain amino acid aminotransferase 4.444 −69 ATTATAATATTCCAAT (SEQ ID NO: 271) — SP1211 — hypothetical protein 4.444 −296 ATTATAATATTCCAAT (SEQ ID NO: 272) — SP1210 — hypothetical protein 4.41439 −134 ATTATTATATAGCAAT (SEQ ID NO: 273) — SP2103 COG0500 rRNA (guanine-N1-)-methyltransferase 4.41439 −1 ATTGCTATATAATAAT (SEQ ID NO: 274) — SP2102 — hypothetical protein 4.41439 −314 ATTGCTATATAATAAT (SEQ ID NO: 275) — SP2101 COG2217 cation-transporting ATPase, EI-E2 family 4.3461 −68 ACTGTTATATAATACT (SEQ ID NO: 276) — SP0088 COG0840 hypothetical protein 4.3461 −19 AGTATTATATAACAGT (SEQ ID NO: 277) — SP0087 — hypothetical protein 4.32609 −290 AGTGGTCTATTCGAAT (SEQ ID NO: 278) — SP1249 COG0516 conserved hypothetical protein 4.27568 −345 CTTGGGATAAACCACT (SEQ ID NO: 279) — SP1702 COG0653 preprotein translocase, SecA subunit 4.27445 −316 ATTAGAATATAAAAAT (SEQ ID NO: 280) — SP1956 — hypothetical protein 4.27389 −93 ATAGGTCTATACCATT (SEQ ID NO: 281) — SP2056 COG1820 N-acetylglucosamine-6-phosphate deacetylase 4.25853 −232 AGTGTTGTATGCCAGT (SEQ ID NO: 282) — SP0056 COG0015 adenylosuccinate lyase 4.25658 −35 TTTGGAGTATTCCAAT (SEQ ID NO: 283) — SP1319 COG1527 v-type sodium ATP synthase, subunit C 4.24921 −212 AAGGATATATACCAAT (SEQ ID NO: 284) — SP1431 COG2189 type II DNA modification methyltransferase, putative 4.24425 −46 AGTGGTATATTTAATT (SEQ ID NO: 285) — SP0474 COG1455 PTS system, cellobiose-specific IIC component 4.24425 −138 AATTAAATATACCACT (SEQ ID NO: 286) — SP0473 COG1940 ROK family protein 4.24051 −42 AATGGTATAATTCATT (SEQ ID NO: 287) — SP1810 — hypothetical protein 4.23862 −94 TATACTATATACCATT (SEQ ID NO: 288) — SP0839 COG1072 pantothenate kinase 4.23862 −112 AATGGTATATAGTATA (SEQ ID NO: 289) — SP0840 — hypothetical protein 4.23664 −76 ATTGGCATATCAGACT (SEQ ID NO: 290) — SP1264 COG1808 conserved domain protein 4.2252 −87 AATGTGATATAATAGT (SEQ ID NO: 291) — SP1421 COG1488 conserved hypothetical protein 4.21304 −40 CTTTGTATATACTAGT (SEQ ID NO: 292) — SP0394 COG2213 PTS system, mannitol-specific IIBC components 4.21243 −149 ATGGGGATATAACATT (SEQ ID NO: 293) — SP2159 — fucolectin-related protein 4.20615 −50 AGTGTGATATAATAGT (SEQ ID NO: 294) — SP0499 COG0126 phosphoglycerate kinase 4.20387 −42 ACTAGTATAGCACAAT (SEQ ID NO: 295) — SP1011 — GtrA family protein B. S. pyogenes 4.95709 −66 AGTGGTATATACCATT (SEQ ID NO: 296) nagA SPy1694 COG1820 putative N-acetylglucosamine-6-phosphate deacetylase 4.76896 −156 TATGGTATATACCAAT (SEQ ID NO: 297) queA SPy1400 COG0809 putative S-adenosylmethionine-tRNA ribosyltransferase-isomerase 4.76896 −62 ATTGGTATATACCATA (SEQ ID NO: 298) nagB SPy1399 COG0363 putative N-acetylglucosamine-6-phosphate isomerase 4.57524 −163 ATTAGCATATCCCAAT (SEQ ID NO: 299) — SPy0433 — hypothetical protein 4.45835 −148 ATTAGACTATACCAAT (SEQ ID NO: 300) glmS SPy1280 COG0449 putative L-glutamine-D-fructose-6-phosphate amidotransferase 4.31846 −123 ATTGTGATATAATAAT (SEQ ID NO: 301) gatC SPy1772 COG0721 putative Glu-tRNA Gln amidotransferase subunit C 4.29471 −2 AATGATATATAATAAT (SEQ ID NO: 302) — SPy0045 COG0534 conserved hypothetical protein 4.2322 −131 AATTGGATATCACAAT (SEQ ID NO: 303) — SPy0593 — conserved hypothetical protein 4.21387 −155 AGTTTAATATCCCAAT (SEQ ID NO: 304) lysS SPy0595 COG1190 putative lysyl-tRNA synthetase 4.21387 −35 ATTGGGATATTAAACT (SEQ ID NO: 305) — SPy0596 COG1011 conserved hypothetical protein 4.21271 −144 TATGGAATATTACACT (SEQ ID NO: 306) hasB SPy2201 COG1004 UDP-glucose 6-dehydrogenase 4.20978 −301 ACTTGTATATGCCAAG (SEQ ID NO: 307) accD SPy1744 COG0777 putative acetyl-CoA carboxylase beta subunit 4.20304 −170 ACTGTTATATAGTATT (SEQ ID NO: 308) acoA SPy1026 COG1071 putative acetoin dehydrogenase (TPP-dependent) alpha chain 4.2014 −69 ATTGTATTATAACAAT (SEQ ID NO: 309) — SPy1884 COG0330 similar to several eukaryotic hypersensitive-induced response proteins 4.17464 −139 AGTGGCATAACACAAT (SEQ ID NO: 310) fabG SPy1749 COG1028 putative beta-ketoacyl-ACP reductase 4.16261 −165 TGTTGGATATTCCAAT (SEQ ID NO: 311) — SPy1253 — conserved hypothetical protein 4.1621 −17 ATTCGGATATAACAAA (SEQ ID NO: 312) — SPy1297 COG1609 putative transcription regulator (LacI family) 4.14754 −111 ATTAGTATAGGCTACT (SEQ ID NO: 313) — SPy1437 — hypothetical protein 4.13534 −279 ATTGGGATATGCAACA (SEQ ID NO: 314) pyrR SPy0830 COG2065 putative pyrimidine regulatory protein 4.12956 −148 AATTGTATAGACCAAC (SEQ ID NO: 315) — SPy0539 — hypothetical gene 4.1271 −161 AATGAAATATTCAAAT (SEQ ID NO: 316) — SPy2099 COG2188 putative transcriptional regulator (GntR family) 4.1271 −66 ATTTGAATATTTCATT (SEQ ID NO: 317) — SPy2097 COG1264 putative PTS system enzyme II 4.12284 −105 ATTGTGCTAGACCATT (SEQ ID NO: 318) — SPy1494 — hypothetical protein 4.10357 −100 ATTGGAATATGATAAA (SEQ ID NO: 319) — SPy1249 COG1393 conserved hypothetical protein 4.10336 −42 AATAGTATATTAGATT (SEQ ID NO: 320) — SPy0338 COG1327 conserved hypothetical protein 4.09424 −271 ATTGGTACATGTCAAT (SEQ ID NO: 321) glpF.2 SPy1854 COG0580 putative glycerol uptake facilitator protein C. S. mutans 4.71268 −42 TTTGGTATATACCATT (SEQ ID NO: 322) — SMU.435 — putative N-acetylglucosamine-6-phosphate deacetylase 4.56555 −17 ATTGGAATACACCAAT (SEQ ID NO: 323) — SMU.284 — hypothetical protein 4.39528 −69 ATTAGACTATACCAAT (SEQ ID NO: 324) glmS SMU.1187 — glucosamine-fructose-6-phosphate aminotransferase 4.35759 −36 ACTGGTATAAACCAAA (SEQ ID NO: 325) gtfA SMU.881 — sucrose phosphorylase, GtfA 4.31505 −125 AATGTTATATTACAGT (SEQ ID NO: 326 — SMU.995 — putative ABC transporter, permease protein; possible ferrichrome transport system 4.28134 −295 AATGGGAAATACCATT (SEQ ID NO: 327) rexA SMU.1499 — putative exonuclease RexA 4.27644 −101 ATTGGAATATAAGACT (SEQ ID NO: 328) — SMU.458 — putative ATP-dependent RNA helicase 4.26492 −195 ATTAGTATAAAACAAT (SEQ ID NO: 329) — SMU.1912c — hypothetical protein 4.23783 −238 ATTGATATATTTCAAT (SEQ ID NO: 330) mleS SMU.137 — malolactic enzyme 4.21286 −67 AATAGTTTATACTAAT (SEQ ID NO: 331) — SMU.753 — conserved hypothetical protein 4.19185 −199 AGTTTTATATAACAAT (SEQ ID NO: 332) — SMU.1145c — putative histidine kinase; homolog of RumK and ScnK 4.14917 −331 TATGGAATATAATAAT (SEQ ID NO: 333) parC SMU.1204 — topoisomerase IV, subunit A 4.14346 −39 AATAGTTTATACTACT (SEQ ID NO: 334) — SMU.1349 — hypothetical protein 4.14346 −330 AGTAGTATAAACTATT (SEQ ID NO: 335) — SMU.1348c — putative ABC transporter, ATP-binding protein 4.11591 −270 ATTGATATAGAACAGT (SEQ ID NO: 336) pstS SMU.1138 — putative ABC transporter, phosphate-binding protein 4.11572 −161 GGTGGAATAGTCCAAT (SEQ ID NO: 337) glgB SMU.1539 — putative 1,4-alpha-glucan branching enzyme 4.10525 −298 TGTGGCCTATGCCAAT (SEQ ID NO: 338) — SMU.166 — hypothetical protein 4.10485 3 AATGGTATAAAAAAAT (SEQ ID NO: 339) msmE SMU.878 — multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE 4.10136 −341 ATTAGAATATGGCAGT (SEQ ID NO: 340) hprT SMU.14 — putative hypoxanthine-guanine phosphoribosyltransferase 4.10001 −125 ATTAGAATATACCTCT (SEQ ID NO: 341) — SMU.1908c — hypothetical protein 4.0932 −38 ATTGGTATATTAAAAA (SEQ ID NO: 342) — SMU.1764c — conserved hypothetical protein 4.0924 −252 AAAGGTATAAACCATT (SEQ ID NO: 343) — SMU.2162c — conserved hypothetical protein 4.09059 −161 TTTAGAATAGACCATT (SEQ ID NO: 344) guaB SMU.2157 — inosine monophosphate dehydrogenase 4.07559 −65 AATTTGATATTCCAGT (SEQ ID NO: 345) rmlC SMU.1460 — putative dTDP-4-keto-L-rhamnose reductase 4.06457 −113 TTTATTATATACTATT (SEQ ID NO: 346) — SMU.624 — putative 1-acylglycerol-3-phosphate O-acyltransferase 4.06457 −42 AATAGTATATAATAAA (SEQ ID NO: 347) — SMU.623c — putative deacetylase 4.05513 −256 AATAGCTTATACTAAT (SEQ ID NO: 348) — SMU.40 — conserved hypothetical protein 4.05348 −42 AGTGTTATATGCTATA (SEQ ID NO: 349) scnR SMU.1815 — putative response regulator; ScnR homolog 4.0529 −215 TGTGGTTTATACCACA (SEQ ID NO: 350) asd SMU.989 — aspartate-semialdehyde dehydrogenase D. S. agalactiae 4.67663 −61 ATTGGTATATACCATA (SEQ ID NO: 351) nagB SAG0799 — glucosamine-6-phosphate isomerase 4.57476 −306 AATGGAATATACTAAT (SEQ ID NO: 352) — SAG0698 — beta-glucuronidase 4.43079 −66 ATAGGTATATACCATT (SEQ ID NO: 353) nagA SAG0266 — N-acetylglucosamine-6-phosphate deacetylase 4.42251 −54 ATTGGTATATATTAAT (SEQ ID NO: 354) — SAG0943 — hypothetical protein 4.42251 −74 ATTAATATATACCAAT (SEQ ID NO: 355) glmS SAG0944 — glucosamine--fructose-6-phosphate aminotransferase, isomerizing 4.37914 −81 AGTGGTATAATCCAGT (SEQ ID NO: 356) ksgA SAG1779 — dimethyladenosine transferase 4.37243 −332 ATTGGTATATATTATT (SEQ ID NO: 357) — SAG1033 — FtsK/SpoIIIE family protein 4.33644 −229 ATTGGAATATCCGATT (SEQ ID NO: 358) — SAG2003 — IS1381, transposase OrfA 4.28704 −61 AATGGTATATCACAAG (SEQ ID NO: 359) — SAG2008 — conserved hypothetical protein 4.20258 −210 TCTATTATATACCAAT (SEQ ID NO: 360) — SAG2170 — conserved hypothetical protein 4.20258 −50 ATTGGTATATAATAGA (SEQ ID NO: 361) — SAG2169 — membrane protein, putative 4.13411 −232 AATATGATATACTAAT (SEQ ID NO: 362) — SAG1186 — metallo-beta-lactamase superfamily protein 4.13259 −292 CTTGGAATATTCCATA (SEQ ID NO: 363) — SAG0699 — transcriptional regulator, GntR family 4.10916 −124 AGTAGAATAGTCCATT (SEQ ID NO: 364) — SAG1951 — PTS system, IIA component, putative 4.09376 −202 AGTGGAATAGACAAGT (SEQ ID NO: 365) cglB SAG0164 — competence protein CglB 4.09142 −270 AGTGGTATAATCCAGG (SEQ ID NO: 366) — SAG1307 — hypothetical protein 4.09022 −9 ATTGGGCTATGCGAAT (SEQ ID NO: 367) — SAG0277 — conserved hypothetical protein 4.08873 −44 ATTAGGATAAACTAAT (SEQ ID NO: 368) — SAG0021 — protease, putative 4.08273 −289 ACTTGAATATCCTAAT (SEQ ID NO: 369) — SAG0626 — MutT/nudix family protein 4.07572 −69 TATAGTATATAGCATT (SEQ ID NO: 370) neuB SAG1161 — N-acetyl neuramic acid synthetase NeuB 4.07402 −31 ATTTTAATATAACAAT (SEQ ID NO: 371) pepX SAG1736 — X-pro dipeptidyl-peptidase 4.07379 −257 AGTTGAATATGCTAAT (SEQ ID NO: 372) — SAG1571 — hypothetical protein 4.06691 −122 ATTGGTATTTACGAGT (SEQ ID NO: 373) — SAG1711 — magnesium transporter, CorA family 4.06603 −120 AATGGAATATTTTATT (SEQ ID NO: 374) cylF SAG0670 — cylF protein 4.04872 −169 CATGGGATATTCAAAT (SEQ ID NO: 375) — SAG1260 — hypothetical protein 4.04872 −324 GTTGGAATATCGCATT (SEQ ID NO: 376) tkt SAG0278 — transketolase 4.04511 −103 ATTGGCTTATTCAAAT (SEQ ID NO: 377) — SAG0231 — hypothetical protein 4.0417 −180 AATGATATATGCAACT (SEQ ID NO: 378) asd SAG1051 — aspartate-semialdehyde dehydrogenase 4.02438 −141 ATTGTCATATAACACC (SEQ ID NO: 379) — SAG1569 — copper homeostasis protein CutC, putative 4.0221 −8 ATTAGTATATGTCAAA (SEQ ID NO: 380) — SAG1683 — immunogenic secreted protein, putative 4.00725 −283 AGTACAATATAACAAT (SEQ ID NO: 381) — SAG1982 — transcriptional regulator, Cro/CI family

TABLE 8 DasR binding sites in Listeria species. Score Position Site Gene Synonym COG Product A. Listeria innocua 5.36566 −121 ATTGGTCTATATCAAT (SEQ ID NO: 382) — lin1996 COG3469 similar to chitinases 5.36389 −38 ATTGGTATAGACCGAT (SEQ ID NO: 383) — lin0955 COG1820 similar to N-acetylglucosamine-6P-phosphate deacetylase (EC 3.5.1.25) 5.26731 −173 AATGGTCTAGACAAAT (SEQ ID NO: 384) codV lin1316 COG0582 similar to integrase/recombinase 5.24253 −34 ACTTGTATATACAAGT (SEQ ID NO: 385) — lin1223 COG1264 similar to PTS system trehalose specific enzyme IIBC 5.24253 −98 ACTTGTATATACAAGT (SEQ ID NO: 386) — lin1224 COG0494 lin1224 5.16643 −44 ACTGGTATAAACAAGT (SEQ ID NO: 387) — lin0296 COG0366 lin0296 5.0365 −143 AACTGTCTAGACCAAT (SEQ ID NO: 388) — lin0780 COG1113 similar to amino acid transporter 4.98056 −87 ATTGGTATAAAGCAGT (SEQ ID NO: 389) — lin2570 — similar to Orf51 [bacteriophage bIL285] 4.97549 −138 ATCGGTTTATACCGGT (SEQ ID NO: 390) — lin1779 COG0803 similar to ABC transporter and adhesion proteins 4.96433 −93 TTTTGTATAGACCAAT (SEQ ID NO: 391) fbp lin0825 COG0639 highly similar to fructose-1,6-bisphosphatase 4.95402 −201 ACAAGTATAGACCAAT (SEQ ID NO: 392) — lin1606 COG0205 lin1606 4.90623 −72 ATTTGTCTATAATAAT (SEQ ID NO: 393) pheT lin1648 COG0073 similar phenylalanyl-tRNA synthetase (B subunit) 4.89907 −315 ACTGTTTTATACAAAT (SEQ ID NO: 394) pflB lin1443 COG1882 pyruvate formate-lyase 4.86345 −1 AATGGTCAATACAAAT (SEQ ID NO: 395) aroA lin1641 COG1605 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 4.83652 −129 AGTGGTCTATATAATT (SEQ ID NO: 396) — lin1069 — lin1069 4.82408 −299 ATCGGTATATACTTGT (SEQ ID NO: 397) — lin1328 — internalin like protein (LPXTG motif) 4.80458 −342 AATGGTTTATATCACT (SEQ ID NO: 398) — lin0432 COG0574 similar to phosphoenolpyruvate synthase 4.79819 −178 AATGCTTTATACAAAT (SEQ ID NO: 399) — lin0169 — similar to phage proteins 4.78736 −349 AATGGAATACACCAAT (SEQ ID NO: 400) — lin0406 — lin0406 4.77453 −294 ACAGATCTAGACCAGT (SEQ ID NO: 401) — lin1754 COG1235 similar to hypothetical protein 44 - Staphylococcus aureus phage phi PVL 4.77453 −295 ACAGATCTAGACCAGT (SEQ ID NO: 402) — lin1243 COG1235 similar to hypothetical protein 44 - Staphylococcus aureus phage phi PVL 4.71781 −330 ATTGGTGTAGATCCGT (SEQ ID NO: 403) — lin1990 COG2759 similar to formyl-tetrahydrofolate synthetase 4.70658 −158 ACTGGTATATATAGCT (SEQ ID NO: 404) — lin2737 COG0489 similar to ATP binding proteins 4.70658 −122 AGCTATATATACCAGT (SEQ ID NO: 405) — lin2738 COG1705 surface protein (GW repeat) similar to N-acetylmuramidase 4.70449 −132 ATTAGTATATAGAATT (SEQ ID NO: 406) — lin2295 COG1393 similar to unknown proteins B. Listeria monocytogenes 5.38367 −121 ATTGGTCTATATCAAT (SEQ ID NO: 407) — lmo1883 COG3469 similar to chitinases 5.37731 −38 ATTGGTATAGACCGAT (SEQ ID NO: 408) — lmo0956 COG1820 similar to N-acetylglucosamine-6P-phosphate deacetylase (EC 3.5.1.25) 5.2828 −47 ATTGGTATAAACAAGT (SEQ ID NO: 409) — lmo0270 — lmo0270 5.25914 −34 ACTTGTATATACAAGT (SEQ ID NO: 410) — lmo1255 COG1264 similar to PTS system trehalose specific enzyme IIBC 5.25914 −98 ACTTGTATATACAAGT (SEQ ID NO: 411) — lmo1256 COG0494 lmo1256 5.07626 −156 ACTTGTATATAACAAT (SEQ ID NO: 412) — lmo1393 COG0612 similar to putative protease 5.05059 −173 AATGGTCTAGACAGAT (SEQ ID NO: 413) codV lmo1277 COG0582 similar to integrase/recombinase 4.97633 −93 TTTTGTATAGACCAAT (SEQ ID NO: 414) fbp lmo0830 COG0639 highly similar to fructose-1,6-bisphosphatase 4.96887 −200 ACAAGTATAGACCAAT (SEQ ID NO: 415) pfk lmo1571 COG0205 highly similar to 6-phosphofructokinase 4.92791 −72 ATTTGTCTATAATAAT (SEQ ID NO: 416) pheT lmo1607 COG0073 similar phenylalanyl-tRNA synthetase (beta subunit) 4.8688 −109 ATTGGTATATACCGGA (SEQ ID NO: 417) — lmo1289 — similar to internalin proteins, putative peptidoglycan bound protein (LPXTG motif) 4.82689 −117 ATTCGTATAGAAAAAT (SEQ ID NO: 418) — lmo1139 — lmo1139 4.82474 −282 AACTGTATATATCAAT (SEQ ID NO: 419) — lmo2445 — similar to internalin 4.79049 −14 AGGGGTCTACACAAGT (SEQ ID NO: 420) — lmo1351 COG0607 lmo1351 4.75049 −81 ATTTGTCGATATCAAT (SEQ ID NO: 421) — lmo2691 COG1705 similar to autolysin, N-acetylmuramidase 4.73024 −181 ATTGGTATAAATTATT (SEQ ID NO: 422) — lmo2748 — similar to B. subtilis stress protein YdaG 4.72697 −234 ACTCGTATATCCAAAT (SEQ ID NO: 423) — lmo0475 — lmo0475 4.72372 −143 AACTATCTAGACCAAT (SEQ ID NO: 424) — lmo0787 COG1113 similar to amino acid transporter 4.71951 −62 ATTAGTATATACTTTT (SEQ ID NO: 425) — lmo2110 COG1482 similar to mannnose-6 phospate isomerase 4.71834 −1 AATGGTTAATACAAAT (SEQ ID NO: 426) aroA lmo1600 COG1605 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 4.71724 −160 ATTTCTGTAGACCATT (SEQ ID NO: 427) — lmo0969 COG0564 similar to ribosomal large subunit pseudouridine synthetase 4.70973 −98 ATGGGAATACACCAAT (SEQ ID NO: 428) — lmo0792 COG0388 similar to conserved hypothetical protein 4.7002 −31 ATCAGTATACACAATT (SEQ ID NO: 429) — lmo1125 — lmo1125 4.69794 −1 AATGATTTATACAATT (SEQ ID NO: 430) fruB lmo2336 COG1105 fructose-1-phosphate kinase 4.69157 −275 ACTTGTTTAAACCGTT (SEQ ID NO: 431) — lmo1219 — lmo1219

TABLE 9 DasR binding sites in Thermobifido fusca. Co-transcribed # Gene Function dre Pos Scr gene Function

Tfu_1418 Tfu_1417c conserved hypothetical protein phospholipid/glycerol acyltransferase AGTGGTCTAGACCTAT (SEQ ID NO: 432) −40 −156 15.0 — — — —

Tfu_3010 Tfu_3009c ketose-bisphosphate aldolase, class-II: Fructose-bisphosphate aldolase, class II, yeast/ E. coli subtype TGTGGTCTAGACCTTT (SEQ ID NO: 433) −155 −222 13.15 Tfu_3011 — conserved hypothetical protein —  3 Tfu_0555 signal transduction histidine kinase ACTGGTCTAGTCCAAT −68 13.03 — — Tfu_0554c dasR, regulatory protein GntR, HTH (SEQ ID NO: 434) −113 — —  4 Tfu_0643 thiamine-monophosphate kinase ACGGGTCTAGACCACT −48 12.88 Tfu_0644 cellulose-binding, family II, (SEQ ID NO: 435) bacterial type Tfu_0642c conserved hypothetical protein −108 — —

Tfu_0136c phosphoserine phosphatase SerB: HAD-superfamily hydrolase subfamily IB, PSPase-like AGTGGCGTAGACCAGG (SEQ ID NO: 436)  23 12.61 — —

Tfu_2555 Tfu_2554c propionyl-CoA carboxylase complex B subunit phosphoenolpyruvate carboxylase ATTGGTCTACTCCACT (SEQ ID NO: 437) −206 −35 12.13 Tfu_2556/ Tfu_2557 — conserved hypothetical protein/ putative acyl-CoA carboxylase, alpha subunit —  7 Tfu_2290 hypothetical protein GCTGGTCTGCACCACG  36 11.72 — — (SEQ ID NO: 438)

Tfu_1037 phosphofructokinase TATGGTCTAGACCATA (SEQ ID NO: 439) −150 11.6 — —

Tfu_0083 phosphoenolpyruvate carboxykinase (GTP) AATGGTCTAGTCCATA (SEQ ID NO: 440) AAAGGTCTAGTCCAAG (SEQ ID NO: 441) −110 −86 11.49 8.92 — — 10 Tfu_0262c hypothetical protein TGTGGTGTCGACCAGC −121 11.4 — — (SEQ ID NO: 442)

Tfu_2611 glmS, glucosamine-fructose- 6-phosphate aminotransferase, isomerising ACTGGTCTATACCGCT (SEQ ID NO: 443) −71 11.24 — —

Tfu_2017c glyceraldehyde-3-phosphate dehydrogenase, type I AAAGGTATAGACCATT (SEQ ID NO: 444) −203 10.59 Tfu_2016 phosphoglycerate kinase 13 Tfu_1774c putative Lsr2-like protein TAAGGTCTATACCTCT −169 10.28 — — (SEQ ID NO: 445) 14 Tfu_1202 putative partitioning or GCAGGTCTACACCCTC −216 10.25 Tfu_1203/ conserved hypothetical protein/ sporulation protein (SEQ ID NO: 446) Tfu_1204/ conserved hypothetical protein/ Tfu_1205 Prokaryotic chromosome segregation and condensation protein ScpB 15 Tfu_2362c putative proteinase CCAGGTGTACACCAGT −201 10.09 — — (SEQ ID NO: 447) 16 Tfu_2741 phosphate ABC transporter, AGGGGTGTACTCCACA −111 10.03 Tfu_2742/ Phosphate transport system permease protein PstC (SEQ ID NO: 448) Tfu_2743 permease protein 2/Phosphate transport system permease protein 1 17 Tfu_2234c putative spermidine synthase TGTGGTGTCGACCATC −2 9.74 — — (SEQ ID NO: 449)

Tfu_0614 D-3-phosphoglycerate dehydrogenase GGTGGTCCACACCAAT (SEQ ID NO:450) −173 9.72 — — 19 Tfu_2626c SecY protein ATTGGTGTGGACCACC −157 9.72 Tfu_2625c/ adenylate kinase, subfamily/ (SEQ ID NO: 451) Tfu_2624c peptidase M24A, methionine aminopeptidase, subfamily 1 20 Tfu_1104 peptidoglycan glycosyltransferase ACTGGACCGCACCACT −52 9.3 — — (SEQ ID NO: 452) 21 Tfu_1818 putative membrane protein CGTGGTG TACACCTAC −276 9.16 — — (SEQ ID NO: 453) 22 Tfu_2283 similar to Cell wall-associated GCTGGCGCAGACCACA −191 9.02 Tfu_2284 hypothetical protein hydrolases (invasion- (SEQ ID NO: 454) associated proteins)

Tfu_0863 pyruvate, phosphate dikinase AGTGGTCTAAATCTCT (SEQ ID NO: 455) ATTGGTTTATACCATT (SEQ ID NO: 456) −230 −134 9.0 8.53 — —

Tfu_1179 pyruvate kinase CTTGGTTTAGACCAAT (SEQ ID NO: 457) −37 8.88 — —

Tfu_0697 Tfu_0696c putative ATP/GTP binding protein putative 6-phosphofructokinase: 1- phosphofructokinase AAAGGTCTAAACCAAT (SEQ ID NO: 458) −306 −116 8.8 — —

Tfu_0433 delta-1-pyrroline-5-carboxylate dehydrogenase 1 ACTGGCCTAGTCCACC (SEQ ID NO: 459) −42 8.69 Tfu_0434/ Tfu_0435 proline dehydrogenase/ conserved hypothetical protein

Tfu_2911c phosphoglycerate mutase 1 AATGGTCTACGCCAAT (SEQ ID NO: 460) −66 8.66 — — 28 Tfu_2361c Tyrosine protein kinase: Serine/ TGTGGGCTGCACCACA −178 8.59 — — threonine protein kinase (SEQ ID NO: 461) 29 Tfu_1504 extracellular solute-binding protein, CGTGGCCTACACCTCC −238 8.54 — — family 3 (SEQ ID NO: 462) 30 Tfu_2622c translation initiation factor IF-1 AGTGATGTACACCACG −306 8.44 — — (SEQ ID NO: 463)

Tfu_0428 Tfu_0427c enolase cell division membrane protein AATGGACTAAACCAAT (SEQ ID NO: 464) −198 −284 8.43 Tfu_0429/ Tfu_0430/ Tfu_0431 — conserved hypothetical protein/ conserved hypothetical protein/ putative hydrolase — 32 Tfu_1002 hedgehog/intein hint, N-terminal AGTGTTCTACGCCATT −297 8.35 — — (SEQ ID NO: 465) 33 Tfu_0600 hypothetical protein AGTGGACTACTCAACG −62 8.18 Tfu_0601 serine/threonine protein kinase (SEQ ID NO: 466) 34 Tfu_2195c trigger factor CGTGGACTGCACAAGT −320 8.17 — — (SEQ ID NO: 467)

Tfu_1033 glucokinase ROK AATGGTTTACTCCATT (SEQ ID NO: 468) −74 8.16 Tfu_1034 conserved hypothetical protein 36 Tfu_1242 putative oxidoreductase CGTGATCTACACCATA −289 8.16 — — (SEQ ID NO: 469)

Tfu_0793 ATPase CGTGGTGGAGTCCACC (SEQ ID NO: 470) −321 8.14 Tfu_0794/ Tfu_0795/ Tfu_0796/ Tfu_0797 helix-turn-helix motif/ Conserved hypothetical protein/CDP-diacylgycerol-- glycerol-3-phosphate 3- phosphatidyltransferase/ CinA, C-terminal 38 Tfu_0213 RNA methyltransferase TrmH, CAAGGACTACGCCACC −181 8.1 — — group 3 (SEQ ID NO: 471) 39 Tfu_0538 molybdenum cofactor CGTGGACTGCGCCACC −289 8.1 Tfu_0539 secreted protein containing a biosynthesis protein E (SEQ ID NO: 472) PDZ domain 40 Tfu_1530c similar to Acetyl/propionyl-CoA GGTGGCGCAGTCCACG −307 8.1 — — carboxylase alpha subunit (SEQ ID NO: 473) 41 Tfu_1691 ABC-type nitrate/sulfonate/ CGAGGTGTACACCAAC −117 8.1 Tfu_1692/ putative ABC transporter bicarbonate transport system (SEQ ID NO: 474) Tfu_1693 membrane protein/ ATPase component putative monooxygenase 42 Tfu_0105c hypothetical protein GCTGGTGCAGTCCATG −247 8.07 — — (SEQ ID NO: 475) 43 Tfu_2986c hypothetical protein GCTGGTCTGCACCGCC −253 8.06 — — (SEQ ID NO: 476) 44 Tfu_0525 conserved hypothetical protein AGTGGTTTCGCCCACT −160 8.05 Tfu_0526/ putative peptidase/conserved (SEQ ID NO: 477) Tfu_0527 hypothetical protein 45 Tfu_2802c putative cytochrome P450 GGGGGTAAAGACCACT −40 8.05 — — (SEQ ID NO: 478) 46 Tfu_2007 6-phosphogluconolactonase GGTGGTGCAGTCCGAT  30 7.98 — — (SEQ ID NO: 479) 47 Tfu_2348 putative ferredoxin reductase ACAGGTGCAGACCATC −4 7.96 — — (SEQ ID NO: 480) Tfu_2347c exonuclease −263 — — 48 Tfu_2320c putative membrane transport protein TGTTGTCTAGAACACA −36 7.95 — — (SEQ ID NO: 481) 49 Tfu_1425 putative integral membrane protein AGAGGTCAACACAATC −159 7.91 — — (SEQ ID NO: 482) Tfu_1424c hypothetical protein −293 — — 50 Tfu_0594 electron transfer flavoprotein, GGTGGTCGAGGCCACC −313 7.88 — — alpha subunit (SEQ ID NO: 483) 51 Tfu_3054c glycosyltransferases involved in GATGGTGAAGACCTCG −81 7.87 — — cell wall biogenesis (SEQ ID NO: 484) 52 Tfu_0527 conserved hypothetical protein CAAGGTCTACTCCACC −216 7.83 — — (SEQ ID NO: 485) 53 Tfu_0658 cell division transporter substrate- TATGGACTACACGATT −1 7.77 — — binding protein FtsY (SEQ ID NO: 486) 54 Tfu_0815c tRNA isopentenyltransferase AGTGGTCCGGACCAAA −247 7.75 — — (SEQ ID NO: 487) 55 Tfu_1226 hypothetical protein CATGGTCTACGCCTCA −296 7.73 Tfu_1227/ putative ferredoxin reductase/ (SEQ ID NO: 488) Tfu_1228/ putative acyl-CoA carboxylase Tfu_1229/ complex A subunit/putative Tfu_1230/ 3-oxoacyl-ACP synthase III/ Tfu_1231 conserved hypothetical protein/ modular polyketide synthase 56 Tfu_2687c NADH-quinone oxidoreductase, AGTGATCCAGACCAGC −318 7.57 Tfu_2686c/ NADH dehydrogenase I chain J/ chain I (SEQ ID NO: 489) Tfu_2685c NADH dehydrogenase I chain K 

1. A method for regulating the expression of a gene of interest in a DasR-like protein comprising micro-organism, comprising providing said micro-organism with a compound capable of interfering with the binding of a DasR-like protein to a DasR-binding site in operable linkage with said gene of interest or a DasR-binding site in operable linkage with a gene whose product is involved in the expression of said gene of interest.
 2. (canceled)
 3. (canceled)
 4. A method for controlling metabolism in a DasR-like protein comprising micro-organism, the method comprising: regulating the binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene whose product is part of a metabolic route
 5. A method according to claim 4, comprising providing to said micro-organism a compound capable of interfering with the binding of said DasR-like protein to said DasR-binding site.
 6. (canceled)
 7. A method according to claim 5, wherein said compound is an inducer of a DasR-regulated metabolic process.
 8. (canceled)
 9. (canceled)
 10. A method for obtaining expression of a product of interest in a DasR-like protein comprising micro-organism, the method comprising: providing said micro-organism with a compound capable of interfering with binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding said product of interest or a DasR-binding site in operable linkage with a gene involved in the production of said product of interest.
 11. A method according to claim 10, wherein the binding of said DasR-like protein to said DasR-binding site is at least in part inhibited by providing said micro-organism with said compound.
 12. A method according to claim 10, wherein the binding of said DasR-like protein to said DasR-binding site is at least in part increased by providing said micro-organism with said compound.
 13. (canceled)
 14. The method according to claim 10, wherein said compound is an inducer of a DasR-regulated process.
 15. The method according to claim 14, wherein said inducer is glucosamine-6-phosphate.
 16. The method according to claim 12, wherein said product of interest is a secondary metabolite.
 17. (canceled)
 18. The method according to claim 12, wherein said product of interest is an enzyme.
 19. (canceled)
 20. A method for at least in part reducing production of an undesired product in a DasR-like protein comprising micro-organism, the method comprising: providing said micro-organism with a compound capable of interfering with binding of a DasR-like protein to a DasR-binding site in operable linkage with a gene encoding said undesired product or a DasR-binding site in operable linkage with a gene involved in the production of undesired product.
 21. A method according to claim 20, wherein said compound is capable of at least in part increasing the binding between said DasR-like protein and said DasR-binding site.
 22. A method according to claim 20, wherein said compound is capable of at least in part decreasing the binding between said DasR-like protein and said DasR-binding site.
 23. The method according to claim 20, wherein the binding of a DasR-like protein to a DasR-binding site is at least in part increased by providing said micro-organism with an increased amount of DasR-binding sites in operable linkage with a gene encoding said undesired product or in operable linkage with a gene involved in the production of said undesired product.
 24. The method according to claim 20, wherein the binding of a DasR-like protein to a DasR-binding site is at least in part decreased by removing at least one DasR binding site in said micro-organism that was originally in operable linkage with a gene encoding said undesired product or in operable linkage with a gene involved in the production of said undesired product.
 25. (canceled)
 26. (canceled)
 27. The method according to claim 1, further comprising providing said micro-organism with a nucleic acid encoding a DasR-like protein.
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. The method according to claim 1, wherein said microorganism is a Streptomyces, a Nocardia, a Thermobifido, an Amycolatopsis, a Planobispora, a Streptoverticillium, a Rhodococcus, or a Corynebacterium.
 32. The method according to claim 1, wherein said micro-organism is a low G+C gram-positive bacterium.
 33. A method according to claim 32, wherein said low G+C gram-positive bacterium is a Bacillus, Lactococcus, Streptococcus, or Listeria.
 34. An activated DasR-repressed cryptic gene cluster, wherein said cryptic gene cluster is activated by decreasing binding of a DasR-like protein to a DasR-binding site in operable linkage with said gene cluster.
 35. An activated cryptic gene cluster according to claim 34, wherein said gene cluster is an antibiotic gene cluster.
 36. The micro-organism comprising an activated cryptic cluster according to claim
 34. 37. A micro-organism comprising a mutated binding site for a DasR-like protein.
 38. A micro-organism comprising a mutant of a DasR-like protein.
 39. (canceled)
 40. A micro-organism comprising increased expression of DasR.
 41. A micro-organism which essentially grows vegetatively and which produces altered levels of a secondary metabolite.
 42. A micro-organism which essentially grows vegetatively and which produces a product from a cryptic gene cluster.
 43. A method for regulating expression of a product of interest in a DasR-like protein comprising micro-organism, the method comprising: providing a nucleic acid encoding said product of interest or a nucleic acid encoding a protein involved in the production of said product of interest with a binding site for a DasR-like protein and wherein said binding site is in operable linkage with said nucleic acid.
 44. A method according to claim 43, wherein said micro-organism further comprises a nucleic acid encoding a DasR-like protein.
 45. (canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. A method for obtaining or improving production of a secondary metabolite in a micro-organism, the method comprising: providing said micro-organism with N-acetylglucosamine or a derivative or a multimer thereof.
 50. (canceled)
 51. (canceled)
 52. An activated DasR-controlled gene cluster, wherein the activated DasR-controlled gene cluster is activated by altering a DasR-like protein's affinity for a DasR-binding site in operable linkage with the activated DasR-controlled gene cluster.
 53. The activated DasR-controlled gene cluster of claim 52, wherein the activated DasR-controlled gene cluster is a cryptic gene cluster. 